Apparatus and method for performing microfluidic manipulations for chemical analysis and synthesis

ABSTRACT

A microchip laboratory system and method provide fluid manipulations for a variety of applications, including sample injection for microchip chemical separations. The microchip is fabricated using standard photolithographic procedures and chemical wet etching, with the substrate and cover plate joined using direct bonding. Capillary electrophoresis and electrochromatography are performed in channels formed in the substrate. Analytes are loaded into a four-way intersection of channels by electrokinetically pumping the analyte through the intersection, followed by switching of the potentials to force an analyte plug into the separation channel.

This is a continuation of U.S. application Ser. No. 09/153,470, filedSep. 15, 1998, now U.S. Pat. No. 6,033,546 which is a continuation ofU.S. application Ser. No. 08/776,645, filed Feb. 3, 1997, now U.S. Pat.No. 5,858,195 which is a continuation-in-part of U.S. application Ser.No. 08/283,769, filed Aug. 1, 1994 now U.S. Pat. No. 6,001,229. Theentire disclosure of the last-mentioned three applications areincorporated by reference in the present specification, as if set forthherein in full.

This invention was made with Government support under contractDE-AC05-84OR21400 awarded by the U.S. Department of Energy to MartinMarietta Energy Systems, Inc. and the Government has certain rights inthis invention.

DESCRIPTION

1. Field of the Invention

The present invention relates generally to miniature instrumentation forchemical analysis, chemical sensing and synthesis and, morespecifically, to electrically controlled manipulations of fluids inmicromachined channels. These manipulations can be used in a variety ofapplications, including the electrically controlled manipulation offluid for capillary electrophoresis, liquid chromatography, flowinjection analysis, and chemical reaction and synthesis.

2. Background of the Invention

Laboratory analysis is a cumbersome process. Acquisition of chemical andbiochemical information requires expensive equipment, specialized labsand highly trained personnel. For this reason, laboratory testing isdone in only a fraction of circumstances where acquisition of chemicalinformation would be useful. A large proportion of testing in bothresearch and clinical situations is done with crude manual methods thatare characterized by high labor costs, high reagent consumption, longturnaround times, relative imprecision and poor reproducibility. Thepractice of techniques such as electrophoresis that are in widespreaduse in biology and medical laboratories have not changed significantlyin thirty years.

Operations that are performed in typical laboratory processes includespecimen preparation, chemical/biochemical conversions, samplefractionation, signal detection and data processing. To accomplish thesetasks, liquids are often measured and dispensed with volumetricaccuracy, mixed together, and subjected to one or several differentphysical or chemical environments that accomplish conversion orfractionation. In research, diagnostic, or development situations, theseoperations are carried out on a macroscopic scale using fluid volumes inthe range of a few microliters to several liters at a time. Individualoperations are performed in series, often using different specializedequipment and instruments for separate steps in the process.Complications, difficulty and expense are often the result of operationsinvolving multiple laboratory processing steps.

Many workers have attempted to solve these problems by creatingintegrated laboratory systems. Conventional robotic devices have beenadapted to perform pipetting, specimen handling, solution mixing, aswell as some fractionation and detection operations. However, thesedevices are highly complicated, very expensive and their operationrequires so much training that their use has been restricted to arelatively small number of research and development programs. Moresuccessful have been automated clinical diagnostic systems for rapidlyand inexpensively performing a small number of applications such asclinical chemistry tests for blood levels of glucose, electrolytes andgases. Unfortunately due to their complexity, large size and great cost,such equipment, is limited in its application to a small number ofdiagnostic circumstances.

The desirability of exploiting the advantages of integrated systems in abroader context of laboratory applications has led to proposals thatsuch systems be miniaturized. In the 1980's, considerable research anddevelopment effort was put into an exploration of the concept ofbiosensors with the hope they might fill the need. Such devices make useof selective chemical systems or biomolecules that are coupled to newmethods of detection such as electrochemistry and optics to transducechemical signals to electrical ones that can be interrupted by computersand other signal processing units. Unfortunately, biosensors have been acommercial disappointment. Fewer than 20 commercialized products wereavailable in 1993, accounting for revenues in the U.S. of less than $100million. Most observers agree that this failure is primarilytechnological rather than reflecting a misinterpretation of marketpotential. In fact, many situations such as massive screening for newdrugs, highly parallel genetic research and testing, micro-chemistry tominimize costly reagent consumption and waste generation, and bedside ordoctor's office diagnostics would greatly benefit from miniatureintegrated laboratory systems.

In the early 1990's, people began to discuss the possibility of creatingminiature versions of conventional technology. Andreas Manz was one ofthe first to articulate the idea in the scientific press. Calling them“miniaturized total analysis systems,” or “μ-TAS,” he predicted that itwould be possible to integrate into single units microscopic versions ofthe various elements necessary to process chemical or biochemicalsamples, thereby achieving automated experimentation. Since that time,miniature components have appeared, particularly molecular separationmethods and microvalves. However, attempts to combine these systems intocompletely integrated systems have not met with success. This isprimarily because precise manipulation of tiny fluid volumes inextremely narrow channels has proven to be a difficult technologicalhurdle.

One prominent field susceptible to miniaturization is capillaryelectrophoresis. Capillary electrophoresis has become a populartechnique for separating charged molecular species in solution. Thetechnique is performed in small capillary tubes to reduce bandbroadening effects due to thermal convection and hence improve resolvingpower. The small tubes imply that minute volumes of materials, on theorder of nanoliters, must be handled to inject the sample into theseparation capillary tube.

Current techniques for injection include electromigration and siphoningof sample from a container into a continuous separation tube. Both ofthese techniques suffer from relatively poor reproducibility, andelectromigration additionally suffers from electrophoreticmobility-based bias. For both sampling techniques the input end of theanalysis capillary tube must be transferred from a buffer reservoir to areservoir holding the sample. Thus, a mechanical manipulation isinvolved. For the siphoning injection, the sample reservoir is raisedabove the buffer reservoir holding the exit end of the capillary for afixed length of time.

An electromigration injection is effected by applying an appropriatelypolarized electrical potential across the capillary tube for a givenduration while the entrance end of the capillary is in the samplereservoir. This can lead to sampling bias because a disproportionatelylarger quantity of the species with higher electrophoretic mobilitiesmigrate into the tube. The capillary is removed from the samplereservoir and replaced into the entrance buffer reservoir after theinjection duration for both techniques.

A continuing need exists for methods and apparatuses which lead toimproved electrophoretic resolution and improved injection stability.

SUMMARY OF THE INVENTION

The present invention provides microchip laboratory systems and methodsthat allow complex biochemical and chemical procedures to be conductedon a microchip under electronic control. The microchip laboratorysystems comprises a material handling apparatus that transportsmaterials through a system of interconnected, integrated channels on amicrochip. The movement of the materials is precisely directed bycontrolling the electric fields produced in the integrated channels. Theprecise control of the movement of such materials enables precisemixing, separation, and reaction as needed to implement a desiredbiochemical or chemical procedure.

The microchip laboratory system of the present invention analyzes and/orsynthesizes chemical material in a precise and reproducible manner. Thesystem includes a body having integrated channels connected a pluralityof reservoirs that store the chemical materials used in the chemicalanalysis or synthesis performed by the system. In one aspect, at leastfive of the reservoirs simultaneously have a controlled electricalpotential, such that material from at least one of the reservoirs istransported through the channels toward at least one of the otherreservoirs. The transportation of the material through the channelsprovides exposure to one or more selected chemical or physicalenvironments, thereby resulting in the synthesis or analysis of thechemical material.

The microchip laboratory system preferably also includes one or moreintersections of integrated channels connecting three or more of thereservoirs. The laboratory system controls the electric fields producedin the channels in a manner that controls which materials in thereservoirs are transported through the intersection(s). In oneembodiment, the microchip laboratory system acts as a mixer or diluterthat combines materials in the intersection(s) by producing anelectrical potential in the intersection that is less than theelectrical potential at each of the two reservoirs from which thematerials to be mixed originate. Alternatively, the laboratory systemcan act as a dispenser that electrokinetically injects precise,controlled amounts of material through the intersection(s).

By simultaneously applying an electrical potential at each of at leastfive reservoirs, the microchip laboratory system can act as a completesystem for performing an entire chemical analysis or synthesis. The fiveor more reservoirs can be configured in a manner that enables theelectrokinetic separation of a sample to be analyzed (“the analyte”)which is then mixed with a reagent from a reagent reservoir.Alternatively, a chemical reaction of an analyte and a solvent can beperformed first, and then the material resulting from the reaction canbe electrokinetically separated. As such, the use of five or morereservoirs provides an integrated laboratory system that can performvirtually any chemical analysis or synthesis.

In yet another aspect of the invention, the microchip laboratory systemincludes a double intersection formed by channels interconnecting atleast six reservoirs. The first intersection can be used to inject aprecisely sized analyte plug into a separation channel toward a wastereservoir. The electrical potential at the second intersection can beselected in a manner that provides additional control over the size ofthe analyte plug. In addition, the electrical potentials can becontrolled in a manner that transports materials from the fifth andsixth reservoirs through the second intersection toward the firstintersection and toward the fourth reservoir after a selected volume ofmaterial from the first intersection is transported through the secondintersection toward the fourth reservoir. Such control can be used topush the analyte plug further down the separation channel while enablinga second analyte plug to be injected through the first intersection.

In another aspect, the microchip laboratory system acts as a microchipflow control system to control the flow of material through anintersection formed by integrated channels connecting at least fourreservoirs. The microchip flow control system simultaneously applies acontrolled electrical potential to at least three of the reservoirs suchthat the volume of material transported from the first reservoir to asecond reservoir through the intersection is selectively controlledsolely by the movement of a material from a third reservoir through theintersection. Preferably, the material moved through the third reservoirto selectively control the material transported from the first reservoiris directed toward the same second reservoir as the material from thefirst reservoir. As such, the microchip flow control system acts as avalve or a gate that selectively controls the volume of materialtransported through the intersection. The microchip flow control systemcan also be configured to act as a dispenser that prevents the firstmaterial from moving through the intersection toward the secondreservoir after a selected volume of the first material has passedthrough the intersection. Alternatively, the microchip flow controlsystem can be configured to act as a diluter that mixes the first andsecond materials in the intersection in a manner that simultaneouslytransports the first and second materials from the intersection towardthe second reservoir.

Other objects, advantages and salient features of the invention willbecome apparent from the following detailed description, which taken inconjunction with the annexed drawings, discloses preferred embodimentsof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of a preferred embodiment of the presentinvention;

FIG. 2 is an enlarged, vertical sectional view of a channel shown in thechannel system of the FIG. 1 embodiment;

FIG. 3 is a schematic, top view of a microchip according to a secondpreferred embodiment of the present invention;

FIG. 4 is an enlarged view of the intersection region of FIG. 3;

FIG. 5 are CCD images of a plug of analyte moving through theintersection of the FIG. 3 embodiment;

FIG. 6 is a schematic top view of a microchip laboratory systemaccording to a third preferred embodiment of a microchip according tothe present invention;

FIG. 7 is a CCD image of “sample loading mode for rhodamine B” (shadedarea);

FIG. 8(a) is a schematic view of the intersection area of the microchipof FIG. 6, prior to analyte injection;

FIG. 8(b) is a CCD fluorescence image taken of the same area depicted inFIG. 8(a), after sample loading in the pinched mode;

FIG. 8(c) is a photomicrograph taken of the same area depicted in FIG.8(a), after sample loading in the floating mode;

FIG. 9 shows integrated fluorescence signals for injected volume plottedversus time for pinched and floating injections;

FIG. 10 is a schematic, top view of a microchip according to a fourthpreferred embodiment of the present invention;

FIG. 11 is an enlarged view of the intersection region of FIG. 10;

FIG. 12 is a schematic top view of a microchip laboratory systemaccording to a fifth preferred embodiment according to the presentinvention;

FIG. 13(a) is a schematic view of a CCD camera view of the intersectionarea of the microchip laboratory system of FIG. 12;

FIG. 13(b) is a CCD fluorescence image taken of the same area depictedin FIG. 13(a), after sample loading in the pinched mode;

FIGS. 13(c)-13(e) are CCD fluorescence images taken of the same areadepicted in FIG. 13(a), sequentially showing a plug of analyte movingaway from the channel intersection at 1, 2, and 3 seconds, respectively,after switching to the run mode;

FIG. 14 shows two injection profiles for didansyl-lysine injected for 2s with γ equal to 0.97 and 9.7;

FIG. 15 are electropherograms taken at (a) 3.3 cm, (b) 9.9 cm, and (c)16.5 cm from the point of injection for rhodamine B (less retained) andsulforhodamine (more retained);

FIG. 16 is a plot of the efficiency data generated from theelectropherograms of FIG. 15, showing variation of the plate number withchannel length for rhodamine B (square with pulse) and sulforhodamine(empty square) with best linear fit (solid lines) for each analyte;

FIG. 17(a) is an electropherogram of rhodamine B and fluorescein with aseparation field strength of 1.5 kV/cm and a separation length of 0.9mm;

FIG. 17(b) is an electropherogram of rhodamine B and fluorescein with aseparation field strength of 1.5 kV/cm and a separation length of 1.6mm;

FIG. 17(c) is an electropherogram of rhodamine B and fluorescein with aseparation field strength of 1.5 kV/cm and a separation length of 11.1mm;

FIG. 18 is a graph showing variation of the number of plates per unittime as a function of the electric field strength for rhodamine B atseparation lengths of 1.6 mm (circle) and 11.1 mm (square) and forfluorescein at separation lengths of 1.6 mm (diamond) and 11.1 mm(triangle);

FIG. 19 shows a chromatogram of coumarins analyzed byelectrochromatography using the system of FIG. 12;

FIG. 20 shows a chromatogram of coumarins resulting from micellarelectrokinetic capillary chromatography using the system of FIG. 12;

FIGS. 21(a) and 21(b) show the separation of three metal ions using thesystem of FIG. 12;

FIG. 22 is a schematic, top plan view of a microchip according to theFIG. 3 embodiment, additionally including a reagent reservoir andreaction channel;

FIG. 23 is a schematic view of the embodiment of FIG. 22, showingapplied voltages;

FIG. 24 shows two electropherograms produced using the FIG. 22embodiment;

FIG. 25 is a schematic view of a microchip laboratory system accordingto a sixth preferred embodiment of the present invention;

FIG. 26 shows the reproducibility of the amount injected for arginineand glycine using the system of FIG. 25;

FIG. 27 shows the overlay of three electrophoretic separations using thesystem of FIG. 25;

FIG. 28 shows a plot of amounts injected versus reaction time using thesystem of FIG. 25;

FIG. 29 shows an electropherogram of restriction fragments producedusing the system of FIG. 30;

FIG. 30 is a schematic view of a microchip laboratory system accordingto a seventh preferred embodiment of the present invention.

FIG. 31 is a schematic view of the apparatus of FIG. 1, showingsequential applications of voltages to effect desired fluidicmanipulations; and

FIG. 32 is a graph showing the different voltages applied to effect thefluidic manipulations of FIG. 31(A)-(C).

DETAILED DESCRIPTION OF THE INVENTION

Integrated, micro-laboratory systems for analyzing or synthesizingchemicals require a precise way of manipulating fluids and fluid-bornematerial and subjecting the fluids to selected chemical or physicalenvironments that produce desired conversions or partitioning. Given theconcentration of analytes that produces chemical conversion inreasonable time scales, the nature of molecular detection, diffusiontimes and manufacturing methods for creating devices on a microscopicscale, miniature integrated micro-laboratory systems lend themselves tochannels having dimensions on the order of 1 to 100 micrometers indiameter. Within this context, electrokinetic pumping has proven to beversatile and effective in transporting materials in microfabricatedlaboratory systems.

The present invention provides the tools necessary to make use ofelectrokinetic pumping not only in separations, but also to performliquid handling that accomplishes other important sample processingsteps, such as chemical conversions or sample partitioning. Bysimultaneously controlling voltage at a plurality of ports connected bychannels in a microchip structure, it is possible to measure anddispense fluids with great precision, mix reagents, incubate reactioncomponents, direct the components towards sites of physical orbiochemical partition, and subject the components to detector systems.By combining these capabilities on a single microchip, one is able tocreate complete, miniature, integrated automated laboratory systems foranalyzing or synthesizing chemicals.

Such integrated micro-laboratory systems can be made up of severalcomponent elements. Component elements can include liquid dispensingsystems, liquid mixing systems, molecular partition systems, detectorsights, etc. For example, as described herein, one can construct arelatively complete system for the identification of restrictionendonuclease sites in a DNA molecule. This single microfabricated devicethus includes in a single system the functions that are traditionallyperformed by a technician employing pipettors, incubators, gelelectrophoresis systems, and data acquisition systems. In this system,DNA is mixed with an enzyme, the mixture is incubated, and a selectedvolume of the reaction mixture is dispensed into a separation channel.Electrophoresis is conducted concurrent with fluorescent labeling of theDNA.

Shown in FIG. 1 is an example of a microchip laboratory system 10configured to implement an entire chemical analysis or synthesis. Thelaboratory system 10 includes six reservoirs 12, 14, 16, 18, 20, and 22connected to each other by a system of channels 24 micromachined into asubstrate or base member (not shown in FIG. 1), as discussed in moredetail below. Each reservoir 12-22 is in fluid communication with acorresponding channel 26, 28, 30, 32, 34 and 36, of the channel system24. The first channel 26 leading from the first reservoir 12 isconnected to the second channel 28 leading from the second reservoir 14at a first intersection 38. Likewise, the third channel 30 from thethird reservoir 16 is connected to the fourth channel 32 at a secondintersection 40. The first intersection 38 is connected to the secondintersection 40 by a reaction chamber or channel 42. The fifth channel34 from the fifth reservoir 20 is also connected to the secondintersection 40 such that the second intersection 40 is a four-wayintersection of channels 30, 32, 34, and 42. The fifth channel 34 alsointersects the sixth channel 36 from the sixth reservoir 22 at a thirdintersection 44.

The materials stored in the reservoirs preferably are transportedelectrokinetically through the channel system 24 in order to implementthe desired analysis or synthesis. To provide such electrokinetictransport, the laboratory system 10 includes a voltage controller 46capable of applying selectable voltage levels, including ground. Such avoltage controller can be implemented using multiple voltage dividersand multiple relays to obtain the selectable voltage levels. The voltagecontroller is connected to an electrode positioned in each of the sixreservoirs 12-22 by voltage lines V1-V6 in order to apply the desiredvoltages to the materials in the reservoirs. Preferably, the voltagecontroller also includes sensor channels S1, S2, and S3 connected to thefirst, second, and third intersections 38, 40, 44, respectively, inorder to sense the voltages present at those intersections.

The use of electrokinetic transport on microminiaturized planar liquidphase separation devices, described above, is a viable approach forsample manipulation and as a pumping mechanism for liquidchromatography. The present invention also entails the use ofelectroosmotic flow to mix various fluids in a controlled andreproducible fashion. When an appropriate fluid is placed in a tube madeof a correspondingly appropriate material, functional groups at thesurface of the tube can ionize. In the case of tubing materials that areterminated in hydroxyl groups, protons will leave the surface and enteran aqueous solvent. Under such conditions the surface will have a netnegative charge and the solvent will have an excess of positive charges,mostly in the charged double layer at the surface. With the applicationof an electric field across the tube, the excess cations in solutionwill be attracted to the cathode, or negative electrode. The movement ofthese positive charges through the tube will drag the solvent with them.The steady state velocity is given by equation 1, $\begin{matrix}{\nu = \frac{ɛ\quad \xi \quad E}{4\pi \quad \eta}} & (1)\end{matrix}$

where v is the solvent velocity, ε is the dielectric constant of thefluid, ξ is the zeta potential of the surface, E is the electric fieldstrength, and n is the solvent viscosity. From equation 1 it is obviousthat the fluid flow velocity or flow rate can be controlled through theelectric field strength. Thus, electroosmosis can be used as aprogrammable pumping mechanism.

The laboratory microchip system 10 shown in FIG. 1 could be used forperforming numerous types of laboratory analysis or synthesis, such asDNA sequencing or analysis, electrochromatography, micellarelectrokinetic capillary chromatography (MECC), inorganic ion analysis,and gradient elution liquid chromatography, as discussed in more detailbelow. The fifth channel 34 typically is used for electrophoretic orelectrochromatographic separations and thus may be referred to incertain embodiments as a separation channel or column. The reactionchamber 42 can be used to mix any two chemicals stored in the first andsecond reservoirs 12, 14. For example, DNA from the first reservoir 12could be mixed with an enzyme from the second reservoir 14 in the firstintersection 38 and the mixture could be incubated in the reactionchamber 42. The incubated mixture could then be transported through thesecond intersection 40 into the separation column 34 for separation. Thesixth reservoir 22 can be used to stored a fluorescent label that ismixed in the third intersection 44 with the materials separated in theseparation column 34. An appropriate detector (D) could then be employedto analyze the labeled materials between the third intersection 44 andthe fifth reservoir 20. By providing for a pre-separation columnreaction in the first intersection 38 and reaction chamber 42 and apost-separation column reaction in the third intersection 44, thelaboratory system 10 can be used to implement many standard laboratorytechniques normally implemented manually in a conventional laboratory.In addition, the elements of the laboratory system 10 could be used tobuild a more complex system to solve more complex laboratory procedures.

The laboratory microchip system 10 includes a substrate or base member(not shown in FIG. 1) which can be an approximately two inch by one inchpiece of microscope slide (Corning, Inc. #2947). While glass is apreferred material, other similar materials may be used, such as fusedsilica, crystalline quartz, fused quartz, plastics, and silicon (if thesurface is treated sufficiently to alter its resistivity). Preferably, anon-conductive material such as glass or fused quartz is used to allowrelatively high electric fields to be applied to electrokineticallytransport materials through channels in the microchip. Semiconductingmaterials such as silicon could also be used, but the electric fieldapplied would normally need to be kept to a minimum (approximately lessthan 300 volts per centimeter using present techniques of providinginsulating layers), which may provide insufficient electrokineticmovement.

The channel pattern 24 is formed in a planar surface of the substrateusing standard photolithographic procedures followed by chemical wetetching. The channel pattern may be transferred onto the substrate witha positive photoresist (Shipley 1811) and an e-beam written chrome mask(Institute of Advanced Manufacturing Sciences, Inc.). The pattern may bechemically etched using HF/NH₄F solution

After forming the channel pattern, a cover plate may then be bonded tothe substrate using a direct bonding technique whereby the substrate andthe cover plate surfaces are first hydrolyzed in a dilute NH₄OH/H₂O₂solution and then joined. The assembly is then annealed at about 500° C.in order to insure proper adhesion of the cover plate to the substrate.

Following bonding of the cover plate, the reservoirs are affixed to thesubstrate, with portions of the cover plate sandwiched therebetween,using epoxy or other suitable means. The reservoirs can be cylindricalwith open opposite axial ends. Typically, electrical contact is made byplacing a platinum wire electrode in each reservoirs. The electrodes areconnected to a voltage controller 46 which applies a desired potentialto select electrodes, in a manner described in more detail below.

A cross section of the first channel is shown in FIG. 2 and is identicalto the cross section of each of the other integrated channels. Whenusing a non-crystalline material (such as glass) for the substrate, andwhen the channels are chemically wet etched, an isotropic etch occurs,i.e., the glass etches uniformly in all directions, and the resultingchannel geometry is trapezoidal. The trapezoidal cross section is due to“undercutting” by the chemical etching process at the edge of thephotoresist. In one embodiment, the channel cross section of theillustrated embodiment has dimensions of 5.2 μm in depth, 57 μm in widthat the top and 45 μm in width at the bottom. In another embodiment, thechannel has a depth “d” of 10 μm, an upper width “w1” of 90 μm, and alower width “w2” of 70 μm.

An important aspect of the present invention is the controlledelectrokinetic transportation of materials through the channel system24. Such controlled electrokinetic transport can be used to dispense aselected amount of material from one of the reservoirs through one ormore intersections of the channel structure 24. Alternatively, as notedabove, selected amounts of materials from two reservoirs can betransported to an intersection where the materials can be mixed indesired concentrations.

Gated Dispenser

Shown in FIG. 3 is a laboratory component 10A that can be used toimplement a preferred method of transporting materials through a channelstructure 24A. The A following each number in FIG. 3 indicates that itcorresponds to an analogous element of FIG. 1 of the same number withoutthe A. For simplicity, the electrodes and the connections to the voltagecontroller that controls the transport of materials through the channelsystem 24A are not shown in FIG. 3.

The microchip laboratory system 10A shown in FIG. 3 controls the amountof material from the first reservoir 12A transported through theintersection 40A toward the fourth reservoir 20A by electrokineticallyopening and closing access to the intersection 40A from the firstchannel 26A. As such, the laboratory microchip system 10A essentiallyimplements a controlled electrokinetic valve. Such an electrokineticvalve can be used as a dispenser to dispense selected volumes of asingle material or as a mixer to mix selected volumes of pluralmaterials in the intersection 40A. In general, electro-osmosis is usedto transport “fluid materials” and electrophoresis is used to transportions without transporting the fluid material surrounding the ions.Accordingly, as used herein, the term “material” is used broadly tocover any form of material, including fluids and ions.

The laboratory system 10A provides a continuous unidirectional flow offluid through the separation channel 34A. This injection or dispensingscheme only requires that the voltage be changed or removed from one (ortwo) reservoirs and allows the fourth reservoir 20A to remain at groundpotential. This will allow injection and separation to be performed witha single polarity power supply.

An enlarged view of the intersection 40A is shown in FIG. 4. Thedirectional arrows indicate the time sequence of the flow profiles atthe intersection 40A. The solid arrows show the initial flow pattern.Voltages at the various reservoirs are adjusted to obtain the describedflow patterns. The initial flow pattern brings a second material fromthe second reservoir 16A at a sufficient rate such that all of the firstmaterial transported from reservoir 12A to the intersection 40A ispushed toward the third reservoir 18A. In general, the potentialdistribution will be such that the highest potential is in the secondreservoir 16A, a slightly lower potential in the first reservoir 12A,and yet a lower potential in the third reservoir 18A, with the fourthreservoir 20A being grounded. Under these conditions, the flow towardsthe fourth reservoir 20A is solely the second material from the secondreservoir 16A.

To dispense material from the first reservoir 12A through theintersection 40A, the potential at the second reservoir 16A can beswitched to a value less than the potential of the first reservoir 12Aor the potentials at reservoirs 16A and/or 18A, can be floatedmomentarily to provide the flow shown by the short dashed arrows in FIG.4. Under these conditions, the primary flow will be from the firstreservoir 12A down towards the separation channel waste reservoir 20A.The flow from the second and third reservoirs 16A, 18A will be small andcould be in either direction. This condition is held long enough totransport a desired amount of material from the first reservoir 12Athrough the intersection 40A and into the separation channel 34A. Aftersufficient time for the desired material to pass through theintersection 40A, the voltage distribution is switched back to theoriginal values to prevent additional material from the first reservoir12A from flowing through the intersection 40A toward the separationchannel 34A.

One application of such a “gated dispenser” is to inject a controlled,variable-sized plug of analyte from the first reservoir 12A forelectrophoretic or chromatographic separation in the separation channel34A. In such a system, the first reservoir 12A stores analyte, thesecond reservoir 16A stores an ionic buffer, the third reservoir 18A isa first waste reservoir and the fourth reservoir 20A is a second wastereservoir. To inject a small variable plug of analyte from the firstreservoir 12A, the potentials at the buffer and first waste reservoirs16A, 18A are simply floated for a short period of time (˜100 ms) toallow the analyte to migrate down the separation column 34A. To breakoff the injection plug, the potentials at the buffer reservoir 16A andthe first waste reservoir 18A are reapplied. Alternatively, the valvingsequence could be effected by bringing reservoirs 16A and 18A to thepotential of the intersection 40A and then returning them to theiroriginal potentials. A shortfall of this method is that the compositionof the injected plug has an electrophoretic mobility bias whereby thefaster migrating compounds are introduced preferentially into theseparation column 34A over slower migrating compounds.

In FIG. 5, a sequential view of a plug of analyte moving through theintersection of the FIG. 3 embodiment can be seen by CCD images Theanalyte being pumped through the laboratory system 10A was rhodamine B(shaded area), and the orientation of the CCD images of the injectioncross or intersection is the same as in FIG. 3. The first image, (A),shows the analyte being pumped through the injection cross orintersection toward the first waste reservoir 18A prior to theinjection. The second image, (B), shows the analyte plug being injectedinto the separation column 34A. The third image, (C), depicts theanalyte plug moving away from the injection intersection after aninjection plug has been completely introduced into the separation column34A. The potentials at the buffer and first waste reservoirs 16A, 18Awere floated for 100 ms while the sample moved into the separationcolumn 34A. By the time of the (C) image, the closed gate mode hasresumed to stop further analyte from moving through the intersection 40Ainto the separation column 34A, and a clean injection plug with a lengthof 142 μm has been introduced into the separation column. As discussedbelow, the gated injector contributes to only a minor fraction of thetotal plate height. The injection plug length (volume) is a function ofthe time of the injection and the electric field strength in the column.The shape of the injected plug is skewed slightly because of thedirectionality of the cleaving buffer flow. However, for a giveninjection period, the reproducibility of the amount injected, determinedby integrating the peak area, is 1% RSD for a series of 10 replicateinjections.

Electrophoresis experiments were conducted using the microchiplaboratory system 10A of FIG. 3, and employed methodology according tothe present invention. Chip dynamics were analyzed using analytefluorescence. A charge coupled device (CCD) camera was used to monitordesignated areas of the chip and a photomultiplier tube (PMT) trackedsingle point events. The CCD (Princeton Instruments, Inc. TE/CCD-512TKM)camera was mounted on a stereo microscope (Nikon SMZ-U), and thelaboratory system 10A was illuminated using an argon ion laser (514.5nm, Coherent Innova 90) operating at 3 W with the beam expanded to acircular spot≈2 cm in diameter. The PMT, with collection optics, wassituated below the microchip with the optical axis perpendicular to themicrochip surface. The laser was operated at approximately 20 mW, andthe beam impinged upon the microchip at a 45° angle from the microchipsurface and parallel to the separation channel. The laser beam and PMTobservation axis were separated by a 135° angle. The point detectionscheme employed a helium-neon laser (543 nm, PMS Electro-opticsLHGP-0051) with an electrometer (Keithley 617) to monitor response ofthe PMT (Oriel 77340). The voltage controller 46 (Spellman CZE 1000R)for electrophoresis was operated between 0 and +4.4 kV relative toground.

The type of gated injector described with respect to FIGS. 3 and 4 showelectrophoretic mobility based bias as do conventional electroosmoticinjections. Nonetheless, this approach has simplicity in voltageswitching requirements and fabrication and provides continuousunidirectional flow through the separation channel. In addition, thegated injector provides a method for valving a variable volume of fluidinto the separation channel 34A in a manner that is precisely controlledby the electrical potentials applied.

Another application of the gated dispenser 10A is to dilute or mixdesired quantities of materials in a controlled manner. To implementsuch a mixing scheme in order to mix the materials from the first andsecond reservoirs 12A, 16A, the potentials in the first and secondchannels 26A, 30A need to be maintained higher than the potential of theintersection 40A during mixing. Such potentials will cause the materialsfrom the first and second reservoirs 12A and 16A to simultaneously movethrough the intersection 40A and thereby mix the two materials. Thepotentials applied at the first and second reservoirs 12A, 16A can beadjusted as desired to achieve the selected concentration of eachmaterial. After dispensing the desired amounts of each material, thepotential at the second reservoir 16A may be increased in a mannersufficient to prevent further material from the first reservoir 12A frombeing transported through the intersection 40A toward the thirdreservoir 30A.

Analyte Injector

Shown in FIG. 6 is a microchip analyte injector 10B according to thepresent invention. The channel pattern 24B has four distinct channels26B, 30B, 32B, and 34B micromachined into a substrate 49 as discussedabove. Each channel has an accompanying reservoir mounted above theterminus of each channel portion, and all four channels intersect at oneend in a four way intersection 40B. The opposite ends of each sectionprovide termini that extend just beyond the peripheral edge of a coverplate 49′ mounted on the substrate 49. The analyte injector 10B shown inFIG. 6 is substantially identical to the gated dispenser 10A except thatthe electrical potentials are applied in a manner that injects a volumeof material from reservoir 16B through the intersection 40B rather thanfrom the reservoir 12B and the volume of material injected is controlledby the size of the intersection.

The embodiment shown in FIG. 6 can be used for various materialmanipulations. In one application, the laboratory system is used toinject an analyte from an analyte reservoir 16B through the intersection40B for separation in the separation channel 34B. The analyte injector10B can be operated in either “load” mode or a “run” mode. Reservoir 16Bis supplied with an analyte and reservoir 12B with buffer. Reservoir 18Bacts as an analyte waste reservoir, and reservoir 20B acts as a wastereservoir.

In the “load” mode, at least two types of analyte introduction arepossible. In the first, known as a “floating” loading, a potential isapplied to the analyte reservoir 16B with reservoir 18B grounded. At thesame time, reservoirs 12B and 20B are floating, meaning that they areneither coupled to the power source, nor grounded.

The second load mode is “pinched” loading mode, wherein potentials aresimultaneously applied at reservoirs 12B, 16B, and 20B, with reservoir18B grounded in order to control the injection plug shape as discussedin more detail below. As used herein, simultaneously controllingelectrical potentials at plural reservoirs means that the electrodes areconnected to a operating power source at the same chemically significanttime period. Floating a reservoir means disconnecting the electrode inthe reservoir from the power source and thus the electrical potential atthe reservoir is not controlled.

In the “run” mode, a potential is applied to the buffer reservoir 12Bwith reservoir 20B grounded and with reservoirs 16B and 18B atapproximately half of the potential of reservoir 12B. During the runmode, the relatively high potential applied to the buffer reservoir 12Bcauses the analyte in the intersection 40B to move toward the wastereservoir 20B in the separation column 34B.

Diagnostic experiments were performed using rhodamine B andsulforhodamine 101 (Exciton Chemical Co., Inc.) as the analyte at 60 μMfor the CCD images and 6 μM for the point detection. A sodiumtetraborate buffer (50 mM, pH 9.2) was the mobile phase in theexperiments. An injection of spatially well defined small volume (≈100pL) and of small longitudinal extent (≈100 μm), injection is beneficialwhen performing these types of analyses.

The analyte is loaded into the injection cross as a frontalelectropherogram, and once the front of the slowest analyte componentpasses through the injection cross or intersection 40B, the analyte isready to be analyzed. In FIG. 7, a CCD image (the area of which isdenoted by the broken line square) displays the flow pattern of theanalyte 54 (shaded area) and the buffer (white area) through the regionof the injection intersection 40B.

By pinching the flow of the analyte, the volume of the analyte plug isstable over time. The slight asymmetry of the plug shape is due to thedifferent electric field strengths in the buffer channel 26B (470 V/cm)and the separation channel 34B (100 V/cm) when 1.0 kV is applied to thebuffer, the analyte and the waste reservoirs, and the analyte wastereservoir is grounded. However, the different field strengths do notinfluence the stability of the analyte plug injected. Ideally, when theanalyte plug is injected into the separation channel 34B, only theanalyte in the injection cross or intersection 40B would migrate intothe separation channel.

The volume of the injection plug in the injection cross is approximately120 pL with a plug length of 130 μm. A portion of the analyte 54 in theanalyte channel 30B and the analyte waste channel 32B is drawn into theseparation channel 34B. Following the switch to the separation (run)mode, the volume of the injection plug is approximately 250 pL with aplug length of 208 μm. These dimensions are estimated from a series ofCCD images taken immediately after the switch is made to the separationmode.

The two modes of loading were tested for the analyte introduction intothe separation channel 34B. The analyte was placed in the analytereservoir 16B, and in both injection schemes was “transported” in thedirection of reservoir 18B, a waste reservoir. CCD images of the twotypes of injections are depicted in FIGS. 8(a)-8(c). FIG. 8(a)schematically shows the intersection 40B, as well as the end portions ofchannels.

The CCD image of FIG. 8(b) is of loading in the pinched mode, just priorto being switched to the run mode. In the pinched mode, analyte (shownas white against the dark background) is pumped electrophoretically andelectroosmotically from reservoir 16B to reservoir 18B (left to right)with buffer from the buffer reservoir 12B (top) and the waste reservoir20B (bottom) traveling toward reservoir 18B (right). The voltagesapplied to reservoirs 12B, 16B, 18B, and 20B were 90%, 90%, 0, and 100%,respectively, of the power supply output which correspond to electricfield strengths in the corresponding channels of 400, 270, 690 and 20V/cm, respectively. Although the voltage applied to the waste reservoir20B is higher than voltage applied to the analyte reservoir 18B, theadditional length of the separation channel 34B compared to the analytechannel 30B provides additional electrical resistance, and thus the flowfrom the analyte buffer 16B into the intersection predominates.Consequently, the analyte in the injection cross or intersection 40B hasa trapezoidal shape and is spatially constricted in the channel 32B bythis material transport pattern.

FIG. 8(c) shows a floating mode loading. The analyte is pumped fromreservoir 16B to 18B as in the pinched injection except no potential isapplied to reservoirs 12B and 20B. By not controlling the flow of mobilephase (buffer) in channel portions 26B and 34B, the analyte is free toexpand into these channels through convective and diffusive flow,thereby resulting in an extended injection plug.

When comparing the pinched and floating injections, the pinchedinjection is superior in three areas: temporal stability of the injectedvolume, the precision of the injected volume, and plug length. When twoor more analytes with vastly different mobilities are to be analyzed, aninjection with temporal stability insures that equal volumes of thefaster and slower moving analytes are introduced into the separationcolumn or channel 34B. The high reproducibility of the injection volumefacilitates the ability to perform quantitative analysis. A smaller pluglength leads to a higher separation efficiency and, consequently, to agreater component capacity for a given instrument and to higher speedseparations.

To determine the temporal stability of each mode, a series of CCDfluorescence images were collected at 1.5 second intervals starting justprior to the analyte reaching the injection intersection 40B. Anestimate of the amount of analyte that is injected was determined byintegrating the fluorescence in the intersection 40B and channels 26Band 34B. This fluorescence is plotted versus time in FIG. 9.

For the pinched injection, the injected volume stabilizes in a fewseconds and has a stability of 1% relative standard deviation (RSD),which is comparable to the stability of the illuminating laser. For thefloating injection, the amount of analyte to be injected into theseparation channel 34B increases with time because of the dispersiveflow of analyte into channels 26B and 34B. For a 30 second injection,the volume of the injection plug is ca. 90 pL and stable for the pinchedinjection versus ca. 300 pL and continuously increasing with time for afloating injection.

By monitoring the separation channel at a point 0.9 cm from theintersection 40B, the reproducibility for the pinched injection mode wastested by integrating the area of the band profile followingintroduction into the separation channel 34B. For six injections with aduration of 40 seconds, the reproducibility for the pinched injection is0.7% RSD. Most of this measured instability is from the opticalmeasurement system. The pinched injection has a higher reproducibilitybecause of the temporal stability of the volume injected. Withelectronically controlled voltage switching, the RSD is expected toimprove for both schemes.

The injection plug width and, ultimately, the resolution betweenanalytes depends largely on both the flow pattern of the analyte and thedimensions of the injection cross or intersection 40B. For this column,the width of the channel at the top is 90 μm, but a channel width of 10μm is feasible which would lead to a decrease in the volume of theinjection plug from 90 pL down to 1 pL with a pinched injection.

There are situations where it may not be desirable to reverse the flowin the separation channel as described above for the “pinched” and“floating” injection schemes. Examples of such cases might be theinjection of a new sample plug before the preceding plug has beencompletely eluted or the use of a post-column reactor where reagent iscontinuously being injected into the end of the separation column. Inthe latter case, it would in general not be desirable to have thereagent flowing back up into the separation channel.

Alternate Analyte Injector

FIG. 10 illustrates an alternate analyte injector system 10C having sixdifferent ports or channels 26C, 30C, 32C, 34C, 56, and 58 respectivelyconnected to six different reservoirs 12C, 16C, 18C, 20C, 60, and 62.The letter C after each element number indicates that the indicatedelement is analogous to a correspondingly numbered elements of FIG. 1.The microchip laboratory system 10C is similar to laboratory systems 10,10A, and 10B described previously, in that an injection cross ofintersection 40C is provided. In the FIG. 10 embodiment, a secondintersection 64 and two additional reservoirs 60 and 62 are alsoprovided to overcome the problems with reversing the flow in theseparation channel.

Like the previous embodiments, the analyte injector system 10C can beused to implement an analyte separation by electrophoresis orchromatography or dispense material into some other processing element.In the laboratory system 10C, the reservoir 12C contains separatingbuffer, reservoir 16C contains the analyte, and reservoirs 18C and 20Care waste reservoirs. Intersection 40C preferably is operated in thepinched mode as in the embodiment shown in FIG. 6. The lowerintersection 64, in fluid communication with reservoirs 60 and 62, areused to provide additional flow so that a continuous buffer stream canbe directed down towards the waste reservoir 20C and, when needed,upwards toward the injection intersection 40C. Reservoir 60 and attachedchannel 56 are not necessary, although they improve performance byreducing band broadening as a plug passes the lower intersection 64. Inmany cases, the flow from reservoir 60 will be symmetric with that fromreservoir 62.

FIG. 11 is an enlarged view of the two intersections 40C and 64. Thedifferent types of arrows show the flow directions at given instances intime for injection of a plug of analyte into the separation channel. Thesolid arrows show the initial flow pattern where the analyte iselectrokinetically pumped into the upper intersection 40C and “pinched”by material flow from reservoirs 12C, 60, and 62 toward this sameintersection. Flow away from the injection intersection 40C is carriedto the analyte waste reservoir 18C. The analyte is also flowing from thereservoir 16C to the analyte waste reservoir 18C. Under theseconditions, flow from reservoir 60 (and reservoir 62) is also going downthe separation channel 34C to the waste reservoir 20C. Such a flowpattern is created by simultaneously controlling the electricalpotentials at all six reservoirs.

A plug of the analyte is injected through the injection intersection 40Cinto the separation channel 34C by switching to the flow profile shownby the short dashed arrows. Buffer flows down from reservoir 12C to theinjection intersection 40C and towards reservoirs 16C, 18C, and 20C.This flow profile also pushes the analyte plug toward waster reservoir20C into the separation channel 34C as described before. This flowprofile is held for a sufficient length of time so as to move theanalyte plug past the lower intersection 64. The flow of buffer fromreservoirs 60 and 62 should be low as indicated by the short arrow andinto the separation channel 34C to minimize distortion.

The distance between the upper and lower intersections 40C and 64,respectively, should be as small as possible to minimize plug distortionand criticality of timing in the switching between the two flowconditions. Electrodes for sensing the electrical potential may also beplaced at the lower intersection and in the channels 56 and 58 to assistin adjusting the electrical potentials for proper flow control. Accurateflow control at the lower intersection 64 may be necessary to preventundesired band broadening.

After the sample plug passes the lower intersection, the potentials areswitched back to the initial conditions to give the original flowprofile as shown with the long dashed arrows. This flow pattern willallow buffer flow into the separation channel 34C while the next analyteplug is being transported to the plug forming region in the upperintersection 40C. This injection scheme will allow a rapid succession ofinjections to be made and may be very important for samples that areslow to migrate or if it takes a long time to achieve a homogeneoussample at the upper intersection 40C such as with entangled polymersolutions. This implementation of the pinched injection also maintainsunidirectional flow through the separation channel as might be requiredfor a post-column reaction as discussed below with respect to FIG. 22.

Serpentine Channel

Another embodiment of the invention is the modofied analyte injectorsystem 10D shown in FIG. 12. The laboratory system 10D shown in FIG. 12is substantially identical to the laboratory system 10B shown in FIG. 6,except that the separation channel 34D follows a serpentine path. Theserpentine path of the separation channel 34D allows the length of theseparation channel to be greatly increased without substantiallyincreasing the area of the substrate 49D needed to implement theserpentine path. Increasing the length of the separation channel 34Dincreases the ability of the laboratory system 10D to distinguishelements of an analyte. In one particularly preferred embodiment, theenclosed length (that which is covered by the cover plate 49D′) of thechannels extending from reservoir 16D to reservoir 18D is 19 mm, whilethe length of channel portion 26D is 6.4 mm and channel 34D is 171 mm.The turn radius of each turn of the channel 34D, which serves as aseparation column, is 0.16 mm.

To perform a separation using the modified analyte injector system 10D,an analyte is first loaded into the injection intersection 40D using oneof the loading methods described above. After the analyte has beenloaded into the intersection 40D of the microchip laboratory system 10,the voltages are manually switched from the loading mode to the run(separation) mode of operation. FIGS. 13(a)-13(e) illustrate aseparation of rhodamine B (less retained) and sulforhodamine (moreretained) using the following conditions: E_(inj)=400 V/cm, E_(run)=150V/cm, buffer=50 mM sodium tetraborate at pH 9.2. The CCD imagesdemonstrate the separation process at 1 second intervals, with FIG.13(a) showing a schematic of the section of the chip imaged, and withFIGS. 13(b)-13(e) showing the separation unfold.

FIG. 13(b) again shows the pinched injection with the applied voltagesat reservoirs 12D, 16D, and 20D equal and reservoir 18D grounded. FIGS.13(c)-13(e) shows the plug moving away from the intersection at 1, 2,and 3 seconds, respectively, after switching to the run mode. In FIG.13(c), the injection plug is migrating around a 90° turn, and banddistortion is visible due to the inner portion of the plug travelingless distance than the outer portion. By FIG. 13(d), the analytes haveseparated into distinct bands, which are distorted in the shape of aparallelogram. In FIG. 13(e), the bands are well separated and haveattained a more rectangular shape, i.e., collapsing of theparallelogram, due to radial diffusion, an additional contribution toefficiency loss.

When the switch is made from the load mode to the run mode, a cleanbreak of the injection plug from the analyte stream is desired to avoidtailing. This is achieved by pumping the mobile phase or buffer fromchannel 26D into channels 30D, 32D, and 34D simultaneously bymaintaining the potential at the intersection 40D below the potential ofreservoir 12D and above the potentials of reservoirs 16D, 18D, and 20D.

In the representative experiments described herein, the intersection 40Dwas maintained at 66% of the potential of reservoir 12D during the runmode. This provided sufficient flow of the analyte back away from theinjection intersection 40D down channels 30D and 32D without decreasingthe field strength in the separation channel 34D significantly.Alternate channel designs would allow a greater fraction of thepotential applied at reservoir 12D to be dropped across the separationchannel 34D, thereby improving efficiency.

This three way flow is demonstrated in FIGS. 13(c)-13(e) as the analytesin channels 30D and 32D (left and right, respectively) move further awayfrom the intersection with time. Three way flow permits well-defined,reproducible injections with minimal bleed of the analyte into theseparation channel 34D.

Detectors

In most applications envisaged for these integrated microsystems forchemical analysis or synthesis it will be necessary to quantify thematerial present in a channel at one or more positions similar toconventional laboratory measurement processes. Techniques typicallyutilized for quantification include, but are not limited to, opticalabsorbance, refractive index changes, fluorescence emission,chemiluminescence, various forms of Raman spectroscopy, electricalconductometric measurements, electrochemical amperiometric measurements,acoustic wave propagation measurements.

Optical absorbence measurements are commonly employed with conventionallaboratory analysis systems because of the generality of the phenomenonin the UV portion of the electromagnetic spectrum. Optical absorbence iscommonly determined by measuring the attenuation of impinging opticalpower as it passes through a known length of material to be quantified.Alternative approaches are possible with laser technology includingphoto acoustic and photo thermal techniques. Such measurements can beutilized with the microchip technology discussed here with theadditional advantage of potentially integrating optical wave guides onmicrofabricated devices. The use of solid-state optical sources such asLEDs and diode lasers with and without frequency conversion elementswould be attractive for reduction of system size. Integration of solidstate optical source and detector technology onto a chip does notpresently appear viable but may one day be of interest.

Refractive index detectors have also been commonly used forquantification of flowing stream chemical analysis systems because ofgenerality of the phenomenon but have typically been less sensitive thanoptical absorption. Laser based implementations of refractive indexdetection could provide adequate sensitivity in some situations and haveadvantages of simplicity. Fluorescence emission (or fluorescencedetection) is an extremely sensitive detection technique and is commonlyemployed for the analysis of biological materials. This approach todetection has much relevance to miniature chemical analysis andsynthesis devices because of the sensitivity of the technique and thesmall volumes that can be manipulated and analyzed (volumes in thepicoliter range are feasible). For example, a 100 pL sample volume with1 nM concentration of analyte would have only 60,000 analyte moleculesto be processed and detected. There are several demonstrations in theliterature of detecting a single molecule in solution by fluorescencedetection. A laser source is often used as the excitation source forultrasensitive measurements but conventional light sources such as raregas discharge lamps and light emitting diodes (LEDs) are also used. Thefluorescence emission can be detected by a photomultiplier tube,photodiode or other light sensor. An array detector such as a chargecoupled device (CCD) detector can be used to image an analyte spatialdistribution.

Raman spectroscopy can be used as a detection method for microchipdevices with the advantage of gaining molecular vibrational information,but with the disadvantage of relatively poor sensitivity. sensitivityhas been increased through surface enhanced Raman spectroscopy (SERS)effects but only at the research level. Electrical or electrochemicaldetection approaches are also of particular interest for implementationon microchip devices due to the ease of integration onto amicrofabricated structure and the potentially high sensitivity that canbe attained. The most general approach to electrical quantification is aconductometric measurement, i.e., a measurement of the conductivity ofan ionic sample. The presence of an ionized analyte can correspondinglyincrease the conductivity of a fluid and thus allow quantification.Amperiometric measurements imply the measurement of the current throughan electrode at a given electrical potential due to the reduction oroxidation of a molecule at the electrode. Some selectivity can beobtained by controlling the potential of the electrode but it isminimal. Amperiometric detection is a less general technique thanconductivity because not all molecules can be reduced or oxidized withinthe limited potentials that can be used with common solvents.Sensitivities in the 1 nM range have been demonstrated in small volumes(10 nL). The other advantage of this technique is that the number ofelectrons measured (through the current) is equal to the number ofmolecules present. The electrodes required for either of these detectionmethods can be included on a microfabricated device through aphotolithographic patterning and metal deposition process. Electrodescould also be used to initiate a chemiluminescence detection process,i.e., an excited state molecule is generated via an oxidation-reductionprocess which then transfers its energy to an analyte molecule,subsequently emitting a photon that is detected.

Acoustic measurements can also be used for quantification of materialsbut have not been widely used to date. One method that has been usedprimarily for gas phase detection is the attenuation or phase shift of asurface acoustic wave (SAW). Adsorption of material to the surface of asubstrate where a SAW is propagating affects the propagationcharacteristics and allows a concentration determination. Selectivesorbents on the surface of the SAW device are often used. Similartechniques may be useful in the devices described herein.

The mixing capabilities of the microchip laboratory systems describedherein lend themselves to detection processes that include the additionof one or more reagents. Derivatization reactions are commonly used inbiochemical assays. For example, amino acids, peptides and proteins arecommonly labeled with dansylating reagents or o-phthaldialdehyde toproduce fluorescent molecules that are easily detectable. Alternatively,an enzyme could be used as a labeling molecule and reagents, includingsubstrate, could be added to provide an enzyme amplified detectionscheme, i.e., the enzyme produces a detectable product. There are manyexamples where such an approach has been used in conventional laboratoryprocedures to enhance detection, either by absorbence or fluorescence. Athird example of a detection method that could benefit from integratedmixing methods is chemiluminescence detection. In these types ofdetection scenarios, a reagent and a catalyst are mixed with anappropriate target molecule to produce an excited state molecule thatemits a detectable photon.

Analyte Stacking

To enhance the sensitivity of the microchip laboratory system 10D, ananalyte pre-concentration can be performed prior to the separation.Concentration enhancement is a valuable tool especially when analyzingenvironmental samples and biological materials, two areas targeted bymicrochip technology. Analyte stacking is a convenient technique toincorporate with electrophoretic analyses. To employ analyte stacking,the analyte is prepared in a buffer with a lower conductivity than theseparation buffer. The difference in conductivity causes the ions in theanalyte to stack at the beginning or end of the analyte plug, therebyresulting in a concentrated analyte plug portion that is detected moreeasily. More elaborate preconcentration techniques include two and threebuffer systems, i.e., transient isotachophoretic preconcentration. Itwill be evident that the greater the number of solutions involved, themore difficult the injection technique is to implement.Pre-concentration steps are well suited for implementation on amicrochip. Electroosmotically driven flow enables separation and samplebuffers to be controlled without the use of valves or pumps. Low deadvolume connections between channels can be easily fabricated enablingfluid manipulation with high precision, speed and reproducibility.

Referring again to FIG. 12, the pre-concentration of the analyte isperformed at the top of the separation channel 34D using a modifiedinjection to stack the analyte. First, an analyte plug is introducedonto the separation channel 34D using electroosmotic flow. The analyteplug is then followed by more separation buffer from the bufferreservoir 16D. At this point, the analyte stacks at the boundaries ofthe analyte and separation buffers. Dansylated amino acids were used asthe analyte, which are anions that stack at the rear boundary of theanalyte buffer plug. Implementation of the analyte stacking is describedalong with the effects of the stacking on both the separation efficiencyand detection limits.

To employ a gated injection using the microchip laboratory system 10D,the analyte is stored in the top reservoir 12D and the buffer is storedin the left reservoir 16D. The gated injection used for the analytestacking is performed on an analyte having an ionic strength that isless than that of the running buffer. Buffer is transported byelectroosmosis from the buffer reservoir 16D towards both the analytewaste and waste reservoirs 18D, 20D. This buffer stream prevents theanalyte from bleeding into the separation channel 34D. Within arepresentative embodiment, the relative potentials at the buffer,analyte, analyte waste and waste reservoirs are 1, 0.9, 0.7 and 0,respectively. For 1 kV applied to the microchip, the field strengths inthe buffer, analyte, analyte waste, and separation channels during theseparation are 170, 130, 180, and 120 V/cm, respectively.

To inject the analyte onto the separation channel 34D, the potential atthe buffer reservoir 16D is floated (opening of the high voltage switch)for a brief period of time (0.1 to 10 s), and analyte migrates into theseparation channel. For 1 kV applied to the microchip, the fieldstrengths in the buffer, sample, sample waste, and separation channelsduring the injection are 0, 240, 120, and 110 V/cm, respectively. Tobreak off the analyte plug, the potential at the buffer reservoir 16D isreapplied (closing of a high voltage switch). The volume of the analyteplug is a function of the injection time, electric field strength, andelectrophoretic mobility.

The separation buffer and analyte compositions can be quite different,yet with the gated injections the integrity of both the analyte andbuffer streams can be alternately maintained in the separation channel34D to perform the stacking operation. The analyte stacking depends onthe relative conductivity of the separation buffer to analyte, γ. Forexample, with a 5 mM separation buffer and a 0.516 mM sample (0.016 mMdansyl-lysine and 0.5 mM sample buffer), γ is equal to 9.7. FIG. 14shows two injection profiles for didansyl-lysine injected for 2 s with γequal to 0.97 and 9.7. The injection profile with γ=0.97 (the separationand sample buffers are both 5 mM) shows no stacking. The second profilewith γ=9.7 shows a modest enhancement of 3.5 for relative peak heightsover the injection with γ=0.97. Didansyl-lysine is an anion, and thusstacks at the rear boundary of the sample buffer plug. In addition toincreasing the analyte concentration, the spatial extent of the plug isconfined. The injection profile with γ=9.7 has a width at half-height of0.41 s, while the injection profile with γ=0.97 has a width athalf-height of 1.88 s. The electric field strength in the separationchannel 34D during the injection (injection field strength) is 95% ofthe electric field strength in the separation channel during theseparation (separation field strength). These profiles are measuredwhile the separation field strength is applied. For an injection time of2 s, an injection plug width of 1.9 s is expected for γ=0.97.

The concentration enhancement due to stacking was evaluated for severalsample plug lengths and relative conductivities of the separation bufferand analyte. The enhancement due to stacking increases with increasingrelative conductivities, γ. In Table 1, the enhancement is listed for gfrom 0.97 to 970. Although the enhancement is largest when γ=970, theseparation efficiency suffers due to an electroosmotic pressureoriginating at the concentration boundary when the relative conductivityis too large. A compromise between the stacking enhancement andseparation efficiency must be reached and γ=10 has been found to beoptimal. For separations performed using stacked injections with γ=97and 970, didansyl-lysine and dansyl-isoleucine could not be resolved dueto a loss in efficiency. Also, because the injection process on themicrochip is computer controlled, and the column is not physicallytransported from vial to vial, the reproducibility of the stackedinjections is 2.1% rsd (percent relative standard deviation) for peakarea for 6 replicate analyses. For comparisor, the non-stacked, gatedinjection has a 1.4% rsd for peak area for 6 replicate analyses, and thepinched injection has a 0.75% rsd for peak area for 6 replicateanalyses. These correspond well to reported values for large-scale,commercial, automated capillary electrophoresis instruments. However,injections made on the microchip are ≈100 times smaller in volume, e.g.100 pL on the microchip versus 10 nL on a commercial instrument.

TABLE 1 Variation of stacking enhancement with relative conductivity, γ.γ Concentration Enhancement 0.97 1 9.7 65 97 11.5 970 13.8

Buffer streams of different conductivities can be accurately combined onmicrochips. Described herein is a simple stacking method, although moreelaborate stacking schemes can be employed by fabricating a microchipwith additional buffer reservoirs. In addition, the leading and trailingelectrolyte buffers can be selected to enhance the sample stacking, andultimately, to lower the detection limits beyond that demonstrated here.It is also noted that much larger enhancements are expected forinorganic (elemental) cations due to the combination of field amplifiedanalyte injection and better matching of analyte and buffer ionmobilities.

Regardless of whether sample stacking is used, the microchip laboratorysystem 10D of FIG. 12 can be employed to achieve electrophoreticseparation of an analyte composed of rhodamine B and sulforhodamine.FIG. 15 are electropherograms at (a) 3.3 cm, (b) 9.9 cm, and (c) 16.5 cmfrom the point of injection for rhodamine B (less retained) andsulforhodamine (more retained). These were taken using the followingconditions: injection type was pinched, E_(inj)=500 V/cm, E_(run)=170V/cm, buffer=50 mM sodium tetraborate at pH 9.2. To obtainelectropherograms in the conventional manner, single point detectionwith the helium-neon laser (green line) was used at different locationsdown the axis of the separation channel 34D.

An important measure of the utility of a separation system is the numberof plates generated per unit time, as given by the formula

N/t=L/(Ht)

where N is the number of theoretical plates, t is the separation time, Lis the length of the separation column, and H is the height equivalentto a theoretical plate. The plate height, H, can be written as

H=A+B/u

where A is the sum of the contributions from the injection plug lengthand the detector path length, B is equal to 2D_(m), where D_(m) is thediffusion coefficient for the analyte in the buffer, and u is the linearvelocity of the analyte.

Combining the two equations above and substituting u=μE where μ is theeffective electrophoretic mobility of the analyte and E is the electricfield strength, the plates per unit time can be expressed as a functionof the electric field strength:

N/t=(μE)²/(AμE+B)

At low electric field strengths when axial diffusion is the dominantform of band dispersion, the term AμE is small relative to B andconsequently, the number of plates per second increases with the squareof the electric field strength.

As the electric field strength increases, the plate height approaches aconstant value, and the plates per unit time increases linearly with theelectric field strength because B is small relative to AμE. It is thusadvantageous to have A as small as possible, a benefit of the pinchedinjection scheme.

The efficiency of the electrophoretic separation of rhodamine B andsulforhodamine at ten evenly spaced positions was monitored, eachconstituting a separate experiment. At 16.5 cm from the point ofinjection, the efficiencies of rhodamine B and sulforhodamine are 38,100and 29,000 plates, respectively. Efficiencies of this magnitude aresufficient for many separation applications. The linearity of the dataprovides information about the uniformity and quality of the channelalong its length. If a defect in the channel, e.g., a large pit, waspresent, a sharp decrease in the efficiency would result; however, nonewas detected. The efficiency data are plotted in FIG. 16 (conditions forFIG. 16 were the same as for FIG. 15).

A similar separation experiment was performed using the microchipanalyte injector 10B of FIG. 6. Because of the straight separationchannel 34B, the analyte injector 10B enables faster separations thanare possible using the serpentine separation channel 34D of thealternate analyte injector 10D shown in FIG. 12. In addition, theelectric field strengths used were higher (470 V/cm and 100 V/cm for thebuffer and separation channels 26B, 34B, respectively), which furtherincreased the speed of the separations.

One particular advantage to the planar microchip laboratory system 10Bof the present invention is that with laser induced fluorescence thepoint of detection can be placed anywhere along the separation column.The electropherograms are detected at separation lengths of 0.9 mm, 1.6mm and 11.1 mm from the injection intersection 40B. The 1.6 mm and 11.1mm separation lengths were used over a range of electric field strengthsfrom 0.06 to 1.5 kV/cm, and the separations had baseline resolution overthis range. At an electric field strength of 1.5 kV/cm, the analytes,rhodamine B and fluorescein, are resolved in less than 150 ms for the0.9 mm separation length, as shown in FIG. 17(a), in less than 260 msfor the 1.6 mm separation length, as shown in FIG. 17(b), and in lessthan 1.6 seconds for the 11.1 mm separation length , as shown in FIG.17(c).

Due to the trapezoidal geometry of the channels, the upper corners makeit difficult to cut the sample plug away precisely when the potentialsare switched from the sample loading mode to the separation mode. Thus,the injection plug has a slight tail associated with it, and this effectprobably accounts for the tailing observed in the separated peaks.

In FIG. 18, the number of plates per second for the 1.6 mm and 11.1 mmseparation lengths are plotted versus the electric field strength. Thenumber of plates per second quickly becomes a linear function of theelectric field strength , because the plate height approaches a constantvalue. The symbols in FIG. 18 represent the experimental data collectedfor the two analytes at the 1.6 mm and 11.1 mm separation lengths. Thelines are calculated using the previously-stated equation and thecoefficients are experimentally determined. A slight deviation is seenbetween the experimental data and the calculated numbers for rhodamine Bat the 11.1 mm separation length. This is primarily due to experimentalerror.

Electrochromatography

A problem with electrophoresis for general analysis is its inability toseparate uncharged species. All neutral species in a particular samplewill have zero electrophoretic mobility, and thus, the same migrationtime. The microchip analyte injector 10D shown in FIG. 12 can also beused to perform electrochromatography to separate non-ionic analytes. Toperform such electrochromatography, the surface of the separationchannel 34D was prepared by chemically bonding a reverse phase coatingto the walls of the separation channel after bonding the cover plate tothe substrate to enclose the channels. The separation channel wastreated with 1 M sodium hydroxide and then rinsed with water. Theseparation channel was dried at 125° C. for 24 hours while purging withhelium at a gauge pressure of approximately 50 kPa. A 25% (w/w) solutionof chlorodimethyloctaldecylsilane (ODS, Aldrich) in toluene was loadedinto the separation channel with an over pressure of helium atapproximately 90 kPa. The ODS/toluene mixture was pumped continuouslyinto the column throughout the 18 hour reaction period at 125° C. Thechannels are rinsed with toluene and then with acetonitrile to removethe unreacted ODS. The laboratory system 10D was used to performelectrochromatography on an analytes composed of coumarin 440 (C440),coumarin 450 (C450) and coumarin 460 (C460; Exciton Chemical Co., Inc.)at 10 μM for the direct fluorescent measurements of the separations and1 μM for the indirect fluorescent measurements of the void time. Asodium tetraborate buffer (10 mM, pH 9.2) with 25% (v/v) acetonitrilewas the buffer.

The analyte injector 10D was operated under a pinched analyte loadingmode and a separation (run) mode as described above with respect to FIG.6. The analyte is loaded into the injection cross via a frontalchromatogram traveling from the analyte reservoir 16D to the analytewaste reservoir 18D, and once the front of the slowest analyte passesthrough the injection intersection 40D, the sample is ready to beanalyzed. To switch to the separation mode, the applied potentials arereconfigured, for instance by manually throwing a switch. Afterswitching the applied potentials, the primary flow path for theseparation is from the buffer reservoir 12D to the waste reservoir 20D.In order to inject a small analyte plug into the separation channel 34Dand to prevent bleeding of the excess analyte into the separationchannel, the analyte and the analyte waste reservoirs 16D, 18D aremaintained at 57% of the potential applied to the buffer reservoir 12D.This method of loading and injecting the sample is time-independent,non-biased and reproducible.

In FIG. 19, a chromatogram of the coumarins is shown for a linearvelocity of 0.65 mm/s. For C440, 11700 plates was observed whichcorresponds to 120 plates/s. The most retained component, C460, has anefficiency nearly an order of magnitude lower than for C440, which was1290 plates. The undulating background in the chromatograms is due tobackground fluorescence from the glass substrate and shows the powerinstability of the laser. This, however, did not hamper the quality ofthe separations or detection. These results compare quite well withconventional laboratory High Performance LC (HPLC) techniques in termsof plate numbers and exceed HPLC in speed by a factor of ten. Efficiencyis decreasing with retention faster than would be predicted by theory.This effect may be due to overloading of the monolayer stationary orkinetic effects due to the high speed of the separation.

Micellar Electrokinetic Capillary Chromatography

In the electrochromatography experiments discussed above with respect toFIG. 19, sample components were separated by their partitioninginteraction with a stationary phase coated on the channel walls. Anothermethod of separating neutral analytes is micellar electrokineticcapillary chromatography (MECC). MECC is an operational mode ofelectrophoresis in which a surfactant such as sodium dodecylsulfate(SDS) is added to the buffer in sufficient concentration to formmicelles in the buffer. In a typical experimental arrangement, themicelles move much more slowly toward the cathode than does thesurrounding buffer solution. The partitioning of solutes between themicelles and the surrounding buffer solution provides a separationmechanism similar to that of liquid chromatography.

The microchip laboratory 10D of FIG. 12 was used to perform on ananalyte composed of neutral dyes coumarin 440 (C440), coumarin 450(C450), and coumarin 460 (C460, Exciton Chemical Co., Inc.). Individualstock solutions of each dye were prepared in methanol, then diluted intothe analysis buffer before use. The concentration of each dye wasapproximately 50 μM unless indicated otherwise. The MECC buffer wascomposed of 10 mM sodium borate (pH 9.1), 50 mM SDS, and 10% (v/v)methanol. The methanol aids in solubilizing the coumarin dyes in theaqueous buffer system and also affects the partitioning of some of thedyes into the micelles. Due care must be used in working with coumarindyes as the chemical, physical, and toxicological properties of thesedyes have not been fully investigated.

The microchip laboratory system 10D was operated in the “pinchedinjection” mode described previously. The voltages applied to thereservoirs are set to either loading mode or a “run” (separation) mode.In the loading mode, a frontal chromatogram of the solution in theanalyte reservoir 16D is pumped electroosmotically through theintersection and into the analyte waste reservoir 18D. Voltages appliedto the buffer and waste reservoirs also cause weak flows into theintersection from the sides, and then into the analyte waste reservoir18D. The chip remains in this mode until the slowest moving component ofthe analyte has passed through the intersection 40D. At this point, theanalyte plug in the intersection is representative of the analytesolution, with no electrokinetic bias.

An injection is made by switching the chip to the “run” mode whichchanges the voltages applied to the reservoirs such that buffer nowflows from the buffer reservoir 12D through the intersection 40D intothe separation channel 34D toward the waste reservoir 20D. The plug ofanalyte that was in the intersection 40D is swept into the separationchannel 34D. Proportionately lower voltages are applied to the analyteand analyte waste reservoirs 16D, 18D to cause a weak flow of bufferfrom the buffer reservoir 12D into these channels. These flows ensurethat the sample plug is cleanly “broken off” from the analyte stream,and that no excess analyte leaks in to the separation channel during theanalysis.

The results of the MECC analysis of a mixture of C440, C450, and C460are shown in FIG. 20. The peaks were identified by individual analysesof each dye. The migration time stability of the first peak, C440, withchanging methanol concentration was a strong indicator that this dye didnot partition into the micelles to a significant extent. Therefore itwas considered an electroosmotic flow marker with migration time t0. Thelast peak, C460, was assumed to be a marker for the micellar migrationtime, tm. Using these values of t0 and tm from the data in FIG. 20, thecalculated elution range, t0/tm, is 0.43. This agrees well with aliterature value of t0/tm=0.4 for a similar buffer system, and supportsour assumption. These results compare well with conventional MECCperformed in capillaries and also shows some advantages over theelectrochromatography experiment described above in that efficiency isretained with retention ratio. Further advantages of this approach toseparating neutral species is that no surface modification of the wallsis necessary and that the stationary phase is continuously refreshedduring experiments.

Inorganic Ion Analysis

Another laboratory analysis that can be performed on either thelaboratory system 10B of FIG. 6 or the laboratory system 10D of FIG. 12is inorganic ion analysis. Using the laboratory system 10B of FIG. 6,inorganic ion analysis was performed on metal ions complexed with8-hydroxyquinoline-5-sulfonic acid (HQS) which are separated byelectrophoresis and detected with UV laser induced fluorescence. HQS hasbeen widely used as a ligand for optical determinations of metal ions.The optical properties and the solubility of HQS in aqueous media haverecently been used for detection of metal ions separated by ionchromatography and capillary electrophoresis. Because uncomplexed HQSdoes not fluoresce, excess ligand is added to the buffer to maintain thecomplexation equilibria during the separation without contributing alarge background signal. This benefits both the efficiency of theseparation and detectability of the sample. The compounds used for theexperiments are zinc sulfate, cadmium nitrate, and aluminum nitrate. Thebuffer is sodium phosphate (60 mM, pH 6.9) with8-hydroxyquinoline-5-sulfonic acid (20 mM for all experiments exceptFIG. 5; Sigma Chemical Co.). AT least 50 mM sodium phosphate buffer isneeded to dissolve up to 20 mM HQS. The substrate 49B used was fusedquartz, which provides greater visibility than glass substrates.

The floating or pinched analyte loading, as described previously withrespect to FIG. 6, is used to transport the analyte to the injectionintersection 40B. With the floating sample loading, the injected plughas no electrophoretic bias, but the volume of sample is a function ofthe sample loading time. Because the sample loading time is inverselyproportional to the field strength used, for high injection fieldstrengths a shorter injection time is used than for low injection fieldstrengths. For example, for an injection field strength of 630 V/cm(FIG. 3a), the injection time is 12 s, and for an injection fieldstrength of 530 V/cm (FIG. 3b), the injection time is 14.5 s. Both thepinched and floating sample loading can be used with and withoutsuppression of the electroosmotic flow.

FIGS. 21(a) and 21(b) show the separation of three metal ions complexedwith 8-hydroxyquinoline-5-sulfonic acid. All three complexes have a netnegative charge. With the electroosmotic flow minimized by the covalentbonding of polyacrylamide to the channel walls, negative potentialsrelative to ground are used to manipulate the complexes during sampleloading and separation. In FIGS. 21(a) and 21(b), the separation channelfield strength is 870 and 720 V/cm, respectively, and the separationlength is 16.5 mm. The volume of the injection plug is 120 pL whichcorresponds to 16, 7 and 19 fmol injected for Zn, Cd, and Al,respectively, for FIG. 21 a. In FIG. 21b, 0.48, 0.23, and 0.59 fmol ofZn, Cd, and Al, respectively, are injected onto the separate column. Theaverage reproducibility of the amounts injected is 1.6% rsd (percentrelative standard deviation) as measured by peak areas (6 replicateanalyses). The stability of the laser used to excite the complexes is≈1% rsd. The detection limits are in a range where useful analyses canbe performed.

Post-Separation Channel Reactor

An alterante microchip laboratory system 10E is shown in FIG. 22. Thefive-port pattern of channels is disposed on a substrate 49E and with acover slip 49E′, as in the previously-described embodiments. Themicrochip laboratory system 10E embodiment was fabricated using standardphotolithographic, wet chemical etching, and bonding techniques. Aphotomask was fabricated by sputtering chrome (50 nm) onto a glass slideand ablating the channel design into the chrome film via a CAD/CAM laserablation system (Resonetics, Inc.). The channel design was thentransferred onto the substrates using a positive photoresist. Thechannels were etched into the substrate in a dilute Hf/Nh₄F bath. Toform the separation channel 34E, a cover -late was bonded to thesubstrate over the etched channels using a direct bonding technique. Thesurfaces were hydrolyzed in dilute NH₄OH/H₂O₂ solution, rinsed indeionized, filtered H₂O, joined and then annealed at 500° C. Cylindricalglass reservoirs were affixed on the substrate using RTV silicone (madeby General Electric). Platinum electrodes provided electrical contactfrom the voltage controller 46E (Spellman CZE1000R) to the solutions inthe reservoirs.

The channel 26E is in one embodiment 2.7 mm in length from the firstreservoir 12E to the intersection 40E, while the channel 30E is 7.0 mm,and the third channel 32E is 6.7 mm. The separation channel 34E ismodified to be only 7.0 mm in length, due to the addition of a reagentreservoir 22E which has a reagent channel 36E that connects to theseparation channel 34E at a mixing tee 44E. Thus, the length of theseparation channel 34E is measured from the intersection 40E to themixing tee 44E. The channel 56 extending from the mixing tee 44E to thewaste reservoir 20E is the reaction column or channel, and in theillustrated embodiment this channel is 10.8 mm in length. The length ofthe reagent channel 36E is 11.6 mm.

In a representative example, the FIG. 22 embodiment was used to separatean analyte and the separation was monitored on-microchip viafluorescence using an argon ion laser (351.1 nm, 50 mW, Coherent Innova90) for excitation. The fluorescence signal was collected with aphotomultiplier tube (PMT, Oriel 77340) for point detection and a chargecoupled device (CCD, Princeton Instruments, Inc. TE/CCD-512TKM) forimaging a region of the microchip. The compounds used for testing theapparatus were rhodamine B (Exciton Chemical Co., Inc.) arginine,glycine, threonine and o-phthaldialdehyde (Sigma Chemical Co.). A sodiumtetraborate buffer (20 mM, pH 9.2) with 2% (v/v) methanol and 0.5% (v/v)β-mercaptoethanol was the buffer in all tests. The concentrations of theamino acid, OPA and rhodamine B solutions were 2 mM, 3.7 mM, and 50 μM,respectively. Several run conditions were utilized.

The schematic view in FIG. 23 demonstrates one example when 1 kV isapplied to the entire system. With this voltage configuration, theelectric field strengths in the separation channel 34E (E_(sep)) and thereaction channel 36E (E_(rxa)) are 200 and 425 V/cm, respectively. Thisallows the combining of 1 part separation effluent with 1.125 partsreagent at the mixing tee 44E. An analyte introduction system such asthis, with or without post-column reaction, allows a very rapid cycletime for multiple analyses.

The electropherograms; (A) and (B) in FIG. 24 demonstrate the separationof two pairs of amino acids. The voltage configuratior is the same as inFIG. 23, except the total applied voltage is 4 kV which corresponds toan electric field strength of 800 V/cm in the separation column(E_(sep)) and 1,700 V/cm. in the reaction column (E_(rxa)). Theinjection times were 100 ms for the tests which correspond to estimatedinjection plug lengths of 384, 245, and 225 μm for arginine, glycine andthreonine, respectively. The injection volumes of 102, 65, and 60 pLcorresponding to 200, 130, and 120 fmol injected for arginine, glycineand threonine, respectively. The point of detection is 6.5 mm downstreamfrom the mixing tee which gives a total column length of 13.5 mm for theseparation and reaction.

The reaction rates of the amino acids with the OPA are moderately fast,but not fast enough on the time scale of these experiments. An increasein the band distortion is observed because the mobilities of thederivatized compounds are different from the pure amino acids. Until thereaction is complete, the zones of unreacted and reacted amino acid willmove at different velocities causing a broadening of the analyte zone.As evidenced in FIG. 24, glycine has the greatest discrepancy inelectrophoretic mobilities between the derivatized and un-derivatizedamino acid. To ensure that the excessive band broadening was not afunction of the retention time, threonine was also tested. Threonine hasa slightly longer retention time than the glycine; however thebroadening is not as extensive as for glycine.

To test the efficiency of the microchip in both the separation columnand the reaction column, a fluorescent laser dye, rhodamine B, was usedas a probe. Efficiency measurements calculated from peak widths at halfheight were made using the point detection scheme at distances of 6 mmand 8 mm from the injection cross, or 1 mm upstream and 1 mm downstreamfrom the mixing tee. This provided information on the effects of themixing of the two streams.

The electric field strengths in the reagent column and the separationcolumn were approximately equal, and the field strength in the reactioncolumn was twice that of the separation column. This configuration ofthe applied voltages allowed an approximately 1:1 volume ratio ofderivatizing reagent and effluent from the separation column. As thefield strengths increased, the degree of turbulence at the mixing teeincreased. At the separation distance of 6 mm (1 mm upstream from themixing tee), the plate height as expected as the inverse of the linearvelocity of the analyte. At the separation distance of 8 mm (1 mmupstream from the mixing tee), the plate height data decreased asexpected as the inverse of the velocity of the analyze. At theseparation distance of 8 mm (1 mm downstream from the mixing tee), theplate height data decreased as expected as the inverse of the velocityof the analyze. At the separation distance of 8 mm (1 mm downstream fromthe mixing tee), the plate height data decreased from 140 V/cm to 280V/cm to 1400 V/cm. This behavior is abnormal and demonstrates a bandbroadening phenomena when two streams of equal volumes converge. Thegeometry of the mixing tee was not optimized to minimize this banddistortion. Above separation field strength of 840 V/cm, the systemstabilizes and again the plate height decreases with increasing linearvelocity. For E_(sep)=1400 V/cm, the ratio of the plate heights at the 8mm and 6 mm separation lengths is 1.22 which is not an unacceptable lossin efficiency for the separation.

The intensity of the fluorescence signal generated from the reaction ofOPA with an amino acid was tested by continuously pumping glycine downthe separation channel to mix with the OPA at the mixing tee. Thefluorescence signal from the OPA/amino acid reaction was collected usinga CCD as the product moved downstream from the mixing tee. Again, therelative volume ratio of the OPA and glycine streams was 1.125. OPA hasa typical half-time of reaction with amino acids of 4 s. The averageresidence times of an analyte molecule in the window of observation are4.68, 2.34, 1.17, and 0.58 s for the electric field strengths in thereaction column (E_(rxm)) of 240, 480, 960, and 1920 V/cm, respectively.The relative intensities of the fluorescence correspond qualitatively tothis 4 s half-time of reaction. As the field strength increases in thereaction channel, the slope and maximum of the intensity of thefluorescence shifts further downstream because the glycine and OPA areswept away from the mixing tee faster with higher field strengths.Ideally, the observed fluorescence from the product would have a stepfunction of a response following the mixing of the separation effluentand derivatizing reagent. However, the kinetics of the reaction and afinite rate of mixing dominated by diffusion prevent this fromoccurring.

The separation using the post-separation channel reactor employed agated injection scheme in order to keep the analyte, buffer and reagentstreams isolated as discussed above with respect to FIG. 3. For thepost-separation channel reactions, the microchip was operated in acontinuous analyte loading/separation mode whereby the analyte wascontinuously pumped from the analyte reservoir 12E through the injectionintersection 40E toward the analyte waste reservoir 18E. Buffer wassimultaneously pumped from the buffer reservoir 16E toward the analytewaste and waste reservoirs 18E, 20E to deflect the analyte stream andprevent the analyte from migrating down the separation channel. Toinject a small aliquot of analyte, the potentials at the buffer andanalyte waste reservoirs 16E, 18E are simply floated for a short periodof time (≈100 ms) to allow the analyte to migrate down the separationchannel as an analyte injection plug. To break off the injection plug,the potentials at the buffer and analyte waste reservoirs 16E, 18E arereapplied.

The use of micromachined post-column reactors can improve the power ofpost-separation channel reactions as an analytical tool by minimizingthe volume of the extra-channel plumbing, especially between theseparation and reagent channels 34E, 36E. This microchip design (FIG.22) was fabricated with modest lengths for the separation channel 34E (7mm) and reagent channel 36E (10.8 mm) which were more than sufficientfor this demonstration. Longer separation channels can be manufacturedon a similar size microchip using a serpentine path to perform moredifficult separations as discussed above with respect to FIG. 12. Todecrease post-mixing tee band distortions, the ratio of the channeldimensions between the separation channel 34E and reaction channel 56should be minimized so that the electric field strength in theseparation channel 34E is large, i.e., narrow channel, and in thereaction channel 56 is small, i.e., wide channel.

For capillary separation systems, the small detection volumes can limitthe number of detection schemes that can be used to extract information.Fluorescence detection remains one of the most sensitive detectiontechniques for capillary electrophoresis. When incorporatingfluorescence detection into a system that does not have naturallyfluorescing analytes, derivatization of the analyte must occur eitherpre- or post-separation. When the fluorescent “tag” is short lived orthe separation is hindered by pre-separation derivatization, post-columnaddition of derivatizing reagent becomes the method of choice. A varietyof post-separation reactors have been demonstrated for capillaryelectrophoresis. However, the ability to construct a post-separationreactor with extremely low volume connections to minimize banddistortion has been difficult. The present invention takes the approachof fabricating a microchip device for electrophoretic separations withan integrated post-separation reaction channel 56 in a single monolithicdevice enabling extremely low volume exchanges between individualchannel functions.

Pre-Separation Channel Reaction System

Instead of the post-separation channel reactor design shown in FIG. 22,the microchip laboratory system 10F shown in FIG. 25 includes spre-separation channel reactor. The pre-separation channel reactordesign shown in FIG. 25 is similar to that shown in FIG. 1, except thatthe first and second channels 26F, 28F form a “goal-post” design withthe reaction chamber 42F rather than the “Y” design of FIG. 1. Thereaction chamber 42F was designed to be wider than the separationchannel 34F to give lower electric field strengths in the reactionchamber and thus longer residence times for the reagents. The reactionchamber is 96 μm wide at half-depth and 6.2 μm deep, and the separationchannel 34F is 31 μm wide at half-depth and 6.2 μm deep.

The microchip laboratory system 10F was used to perform on-linepre-separation channel reactions coupled with electrophoretic analysisof the reaction products. Here, the reactor is operated continuouslywith small aliquots introduced periodically into the separation channel34F using the gated dispenser discussed above with respect to FIG. 3.The operation of the microchip consists of three elements: thederivatization of amino acids with o-phthaldialdehyde (OPA), injectionof the sample onto the separation column, and the separation/ detectionof the components of the reactor effluent. The compounds used for theexperiments were arginine (0.48 mM), glycine (0.58 mM), and OPA (5.1 mM;Sigma Chemical Co.). the buffer in all of the reservoirs was 20 mMsodium tetraborate with 2% (v/v) methanol and 0.5% 2-mercaptoethanol.2-mercaptoethanol is added to the buffer as a reducing agent for thederivatization reaction.

To implement the reaction the reservoirs 12F, 14F, 16F, 18F, and 20Fwere simultaneously given controlled voltages of 0.5 HV, 0.5 HV, HV, 2HV, and ground, respectively. This configuration allowed the lowestpotential drop across the reaction chamber 42F (25 V/cm for 1.0 kVapplied to the microchip) without significant bleeding of the productinto the separation channel when using the gated injection scheme. Thevoltage divider used to establish the potentials applied to each of thereservoirs had a total resistance of 100 MΩ with 10 MΩ divisions. Theanalyte from the first reservoir 12F and the reagent from the secondreservoir 14F are electroosmotically pumped into the reaction chamber42F with a volumetric ratio of 1:1.05. Therefore, the solutions from theanalyte and reagent reservoirs 12F, 14F are diluted by a factor of ≈2.Buffer was simultaneously pumped by electroosmosis from the bufferreservoir 16F toward the analyte waste and waste reservoirs 18F, 20F.This buffer stream prevents the newly formed product from bleeding intothe separation channel 34F.

Preferably, a gated injection scheme, described above with respect toFIG. 3, is used to inject effluent from the reaction chamber 42F intothe separation channel 34F. The potential at the buffer reservoir 16F issimply floated for a brief period of time (0.1 to 1.0 s), and samplemigrates into the separation channel 34F. To break off the injectionplug, the potential at the buffer reservoir 16F is reapplied. The lengthof the injection plug is a function of both the time of the injectionand the electric field strength. With this configuration of appliedpotentials, the reaction of the amino acids with the OPA continuouslygenerates fresh product to be analyzed.

A significant shortcoming of many capillary electrophoresis experimentshas been the poor reproducibility of the injections. Here, because themicrochip injection process is computer controlled, and the injectionprocess involves the opening of a single high voltage switch, theinjections can be accurately time events. FIG. 26 shows thereproducibility of the amount injected (percent relative standarddeviation, % rsd, for the integrated areas of the peaks) for botharginine and glycine at injection field strengths of 0.6 and 1.2 kV/cmand injection times ranging from 0.1 to 1.0 s. For injection timesgreater than 0.3 s, the percent relative standard deviation is below1.8%. This is comparable to reported values for commercial, automatedcapillary electrophoresis instruments. However, injections made on themicrochip are ≈100 times smaller in volume, e.g. 100 pL on the microchipversus 10 nL on a commercial instrument. Part of this fluctuation is dueto the stability of the laser which is ≈0.6%. For injection times>0.3 s,the error appears to be independent of the compound injected and theinjection field strength.

FIG. 27 shows the overlay of three electrophoretic separations ofarginine and glycine after on-microchip pre-column derivatization withOPA with a separation field strength of 1.8 kV/cm and a separationlength of 10 mm. The separation field strength is the electric fieldstrength in the separation channel 34F during the separation. The fieldstrength in the reaction chamber 42F is 150 V/cm. The reaction times forthe analytes is inversely related to their mobilities, e.g., forarginine the reaction time is 4.1 s and for glycine the reaction time is8.9 s. The volumes of the injected plugs were 150 and 71 pL for arginineand glycine, respectively, which correspond to 35 and 20 fmol of theamino acids injected onto the separation channel 34F. The gated injectorallows rapid sequential injections to be made. In this particular case,an analysis could be performed every 4 s. The observed electrophoreticmobilities for the compounds are determined by a linear fit to thevariation of the linear velocity with the separation field strength. Theslopes were 29.1 and 13.3 mm²/(kV-as) for arginine and glycine,respectively. No evidence of Joule heating was observed as indicated bythe linearity of the velocity versus field strength data. A linear fitproduced correlation coefficients of 0.999 for arginine and 0.996 forglycine for separation field strengths from 0.2 to 2.0 kV/cm.

With increasing potentials applied to the microchip laboratory system10F, the field strengths in the reaction chamber 42F and separationchannel 34F increase. This leads to shorter residence times of thereactants in the reaction chamber and faster analysis times for theproducts. By varying the potentials applied to the microchip, thereaction kinetics can be studied. The variation in amount of productgenerated with reaction time is plotted in FIG. 28. The response is theintegrated area of the peak corrected for the residence time in thedetector observation window and photobleaching of the product. Theoffset between the data for the arginine and the glycine in FIG. 28 isdue primarily to the difference in the amounts injected, i.e. differentelectrophoretic mobilities, for the amino acids. A ten-fold excess ofOPA was used to obtain pseudo-first order reaction conditions. Theslopes of the lines fitted to the data correspond to the rates of thederivatization reaction. The slopes are 0.13 s⁻¹ for arginine and 0.11s⁻¹ for glycine corresponding to half-times of reaction of 5.1 and 6.2s, respectively. These half-times of reaction are comparable to the 4 spreviously reported for alanine. We have found no previously reporteddata for arginine or glycine.

These results show the potential power of integrated microfabricatedsystems for performing chemical procedures. The data presented in FIG.28 can be produced under computer control within five approximately fiveminutes consuming on the order of 100 nL of reagents. These results areunprecedented in terms of automation, speed and volume for chemicalreactions.

DNA Analysis

To demonstrate a useful biological analysis procedure, a restrictiondigestion and electrophoretic sizing experiment are performedsequentially on the integrated biochemical reactor/electrophoresismicrochip system 10G shown in FIG. 30. The microchip laboratory system10G is identical to the laboratory system shown in FIG. 25 except thatthe separation channel 34G of the laboratory system 10G follows aserpentine path. The sequence for plasmid pBR322 anc the recognitionsequence of the enzyme Hinf I are known. After digestion, determinationof the fragment distribution is performed by separating the digestionproducts using electrophoresis in a sieving medium in the separationchannel 34 G. For these experiments, hydroxyethyl cellulose is used asthe sieving medium. At a fixed point downstream in the separationchannel 34 G, migrating fragments are interrogated using on-chip laserinduced fluorescence with an intercalating dye, thiazole orange dimer(TOTO-1), as the fluorophore.

The reaction chamber 42 G and separation channel 34 G shown in FIG. 30are 1 and 67 mm long, respectively, having a width at half-depth of 60μm and a depth of 12 μm. In addition, the channel walls are coated withpolyacrylamide to minimize electroosmotic flow and adsorption.Electropherograms are generated using single point detection laserinduced fluorescence detection. An argon ion laser (10 mW) is focused toa spot onto the chip using a lens (100 mm focal length). Thefluorescence signal is collected using a 21 x objective lens(N.A.=0.042), followed by spatial filtering (0.6 mm diameter pinhole)and spectral filtering (560 nm bandpass, 40 nm bandwidth), and measuredusing a photomultiplier tube (PMT). The data acquisition and voltageswitching apparatus are computer controlled. The reaction buffer is 10mM Tris-acetate, 10 mM magnesium acetate, and 50 mM potassium acetate.The reaction buffer is placed in the DNA, enzyme and waste 1 reservoirs12 G, 14 G, 18 G shown in FIG. 30. The separation buffer is 9 mMTris-borate with 0.2 mM EDTA and 1% (w/v) hydroxyethyl cellulose. Theseparation buffer is placed in the buffer and waste 2 reservoirs 16 F,20 F. The concentrations of the plasmid pBR322 and enzyme Hinf I are 125ng/μl and 4 units/μl, respectively. The digestions and separations areperformed at room temperature (20° C.).

The DNA and enzyme are electrophoretically loaded into the reactionchamber 42 G from their respective reservoirs 12 G, 14 G by applicationof proper electrical potentials. The relative potentials at the DNA (12G), enzyme (14 G), buffer (16 G), waste 1 (18 G), and waste 2 (20 G)reservoirs are 10%, 10% 0, 30%, and 100%, respectively. Due to theelectrophoretic mobility differences between the DNA and enzyme, theloading period is made sufficiently long to reach equilibrium. Also, dueto the small volume of the reaction chamber 42 G, 0.7 nL, rapiddiffusional mixing occurs. The electroosmotic flow is minimized by thecovalent immobilization of linear polyacrylamide, thus only anionsmigrate from the DNA and enzyme reservoirs 12 G, 14 G into the reactionchamber 42 G with the potential distributions used. The reaction bufferwhich contains cations, required for the enzymatic digestions, e.g.Mg²⁺, is also placed in the waste 1 reservoir 18 G. This enables thecations to propagate into the reaction chamber countercurrent to the DNAand enzyme during the loading of the reaction chamber. The digestion isperformed statically by removing all electrical potentials after loadingthe reaction chamber 42 G due to the relatively short transit time ofthe DNA through the reaction chamber.

Following the digestion period, the products are migrated into theseparation channel 34 F for analysis by floating the voltages to thebuffer and waste 1 reservoirs 16 G, 18 G. The injection has a mobilitybias where the smaller fragments are injected in favor of the largerfragments. In these experiments the injection plug length for the75-base pair (bp) fragment is estimated to be 0.34 mm whereas for the1632-bp fragment only 0.22 mm. These plug lengths correspond to 34% and22% of the reaction chamber volume, respectively. The entire contents ofthe reaction chamber 42 G cannot be analyzed under current separationconditions because the contribution of the injection plug length to theplate height would be overwhelming.

Following digestion and injection onto the separation channel 34 G, thefragments are resolved using 1.0% (w/v) hydroxyethyl cellulose as thesieving medium. FIG. 29 shows an electropherogram of the restrictionfragments of the plasmid pBR322 following a 2 min digestion by theenzyme Hinf I. To enable efficient on-column staining of thedouble-stranded DNA after digestion but prior to interrogation, theintercalating dye, TOTO-1 (1 μM), is placed in the waste 2 reservoir 20G only and migrates countercurrent to the DNA. As expected, the relativeintensity of the bands increases with increasing fragment size becausemore intercalation sites exist in the larger fragments. The unresolved220/221 and 507/511-bp fragments having higher intensities than adjacentsingle fragment peaks due to the band overlap. The reproducibility ofthe migration times and injection volumes are 0.55 and 3.1 % relativestandard deviation (% rsd), respectively, for 5 replicate analyses.

This demonstration of a microchip laboratory system 10 G that performsplasmid DNA restriction fragment analysis indicates the possibility ofautomating and miniaturizing more sophisticated biochemical procedures.This experiment represents the most sophisticated integrated microchipchemical analysis device demonstrated to date. The device mixes areagent with an analyte, incubates the analyte/reagent mixture, labelsthe products, and analyzes the products entirely under computer controlwhile consuming 10,000 times less material than the typical small volumelaboratory procedure.

In general, the present invention can be used to mix different fluidscontained in different ports or reservoirs. This could be used for aliquid chromatography separation experiment followed by post-columnlabeling reactions in which different chemical solutions of a givenvolume are pumped into the primary separation channel and other reagentsor solutions can be injected or pumped into the stream at differenttimes to be mixed in precise and known concentrations. To execute thisprocess, it is necessary to accurately control and manipulate solutionsin the various channels.

Pre-/Post-Separation Reactor System

FIG. 31 shows the same six port microchip laboratory system 10 shown inFIG. 1, which could take advantage of this novel mixing scheme.Particular features attached to the different ports represent solventreservoirs. This laboratory system could potentially be used for aliquid chromatography separation experiment followed by post-columnlabeling reactions. In such an experiment, reservoirs 12 and 14 wouldcontain solvents to be used in a liquid chromatography solventprogramming type of separation, e.g., water and acetonitrile.

The channel 34 connected to the waste reservoir 2) and to the twochannels 26 and 28 connecting the analyte and solvent reservoirs 12 and14 is the primary separation channel, i.e., where the liquidchromatography experiment would take place. The intersecting channels30, 32 connecting the buffer and analyte waste reservoirs 16 and 18 areused to make an injection into the liquid chromatography or separationchannel 34 as discussed above. Finally, reservoir 22 and its channel 36attaching to the separation channel 34 are used to add a reagent, whichis added in proportions to render the species separated in theseparation channel detectable.

To execute this process, it is necessary to accurately control andmanipulate solutions in the various channels. The embodiments describedabove took very small volume of solution (≈100 pl) from reservoirs 12and 40 and accurately injected them into the separation channel 34. Forthese various scenarios, a given volume of solution needs to betransferred from one channel to another. For example, solventprogramming for liquid chromatography or reagent addition forpost-column labeling reactions requires that streams of solutions bemixed in precise and known concentrations.

The mixing of various solvents in known proportions can be doneaccording to the present invention by controlling potentials whichultimately control electroosmotic flows as indicated in equation 1.According to equation 1 the electric field strength needs to be known todetermine the linear velocity of the solvent. In general, in these typesof fluidic manipulations a known potential or voltage is applied to agiven reservoir. The field strength can be calculated from the appliedvoltage and the characteristics of the channel. In addition, theresistance or conductance of the fluid in the channels must also beknown.

The resistance of a channel is given by equation 2 where R is theresistance, O is the resistivity, L is the length of the channel, and Ais the cross-sectional area. $\begin{matrix}R_{i = \frac{\rho_{i}L_{i}}{A_{i}}} & (2)\end{matrix}$

Fluids are usually characterized by conductance which is just thereciprocal of the resistance as shown in equation 3. In equation 3, K isthe electrical conductance, K is the conductivity, A is thecross-sectional area, and L is the length as above. $\begin{matrix}K_{i = \frac{\kappa_{i}A_{i}}{L_{i}}} & (3)\end{matrix}$

Using ohms law and equations 2 and 3 we can write the field strength ina given channel, i, in terms of the voltage drop across that channeldivided by its length which is equal to the current, I_(i) throughchannel i times the resistivity of that channel divided by thecross-sectional area as shown in equation 4. $\begin{matrix}{{Ei} =_{\begin{matrix}V_{i} \\L_{i}\end{matrix} = {\begin{matrix}{I_{i}P_{i}} \\A_{i}\end{matrix} = \begin{matrix}I_{i} \\{\kappa_{i}A_{i}}\end{matrix}}}} & (4)\end{matrix}$

Thus, if the channel is both dimensionally and electricallycharacterized, the voltage drop across the channel or the currentthrough the channel can be used to determine the solvent velocity orflow rate through that channel as,: expressed in equation 5. It is alsonoted that fluid flow depends on the zeta potential of the surface andthus on the chemical make-ups of the fluid and surface.

V_(i)∝I_(i)∝Flow

Obviously the conductivity, κ, or the resistivity, ρ, will depend uponthe characteristics of the solution which could vary from channel tochannel. In many CE applications the characteristics of the buffer willdominate the electrical characteristics of the fluid, and thus theconductance will be constant. In the case of liquid chromatography wheresolvent programming is performed, the electrical characteristics of thetwo mobile phases could differ considerably if a buffer is not used.During a solvent programming run where the mole fraction of the mixtureis changing, the conductivity of the mixture may change in a nonlinearfashion but it will change monotonically from the conductivity of theone neat solvent to the other. The actual variation of the conductancewith mole fraction depends on the dissociation constant of the solventin addition to the conductivity of the individual ions.

As described above, the device shown schematically in FIG. 31 could beused for performing gradient elution liquid chromatography withpost-column labeling for detection purposes, for example. FIGS. 31(a),31(b), and 31(c) show the fluid flow requirements for carrying out thetask involved in a liquid chromatography experiment as mentioned above.The arrows in the figures show the direction and relative magnitude ofthe flow in the channels. In FIG. 31(a), a volume of analyte from theanalyte reservoir 16 is loaded into the separation intersection 40. Toexecute a pinched injection it is necessary to transport the sample fromthe analyte reservoir 16 across the intersection to the analyte wastereservoir 18. In addition, to confine the lanalyte volume, material fromthe separation channel 34 and the solvent reservoirs 12, 14 must flowtowards the intersection 40 as shown. The flow from the first reservoir12 is much larger than that from the second reservoir 14 because theseare the initial conditions for a gradient elution experiment. At thebeginning of the gradient elution experiment, it is desirable to preventthe reagent in the reagent reservoir 22 from entering the separationchannel 34. To prevent such reagent flow, a small flow of buffer fromthe waste reservoir 20 directed toward the reagent channel 36 isdesirable and this flow should be as near to zero as possible. After arepresentative analyte volume is presented at the injection intersection40, the separation can proceed.

In FIG. 31(b), the run (separation) mode is shown, solvents fromreservoirs 12 and 14 flow through the intersection 40 and down theseparation channel 34. In addition, the solvents flow towards reservoirs4 and 5 to make a clean injection of the analyte into the separationchannel 34. Appropriate flow of reagent from the reagent reservoir 22 isalso directed towards the separation channel. The initial condition asshown in FIG. 31(b) is with a large mole fraction of solvent 1 and asmall mole fraction of solvent 2. The voltages applied to the solventreservoirs 12, 14 are changed as a function of time so that theproportions of solvents 1 and 2 are changed from a dominance of solvent1 to mostly solvent 2. This is shown in FIG. 31(c). The latter monotonicchange in applied voltage effects the gradient elution liquidchromatography experiment. As the isolated component pass the reagentaddition channel 36, appropriate reaction can take place between thisreagent and the isolated material to form a detectable species.

FIG. 32 shows how the voltages to the various reservoirs are changed fora hypothetical gradient elution experiment. The voltages shown in thisdiagram only indicate relative magnitudes and not absolute voltages. Inthe loading mode of operation, static voltages are applied to thevarious reservoirs. Solvent flow from all reservoirs except the reagentreservoir 22 is towards the analyte waste reservoir 18. Thus, theanalyte reservoir 18 is at the lowest potential and all the otherreservoirs are at higher potential. The potential at the reagentreservoir should be sufficiently below that of the waste reservoir 20 toprovide only a slight flow towards the reagent reservoir. The voltage atthe second solvent reservoir 14 should be sufficiently great inmagnitude to provide a net flow towards the injection intersection 40,but the flow should be a low magnitude.

In moving to the run (start) mode depicted in FIG. 31(b), the potentialsare readjusted as indicated in FIG. 32. The flow now is such that thesolvent from the solvents reservoirs 12 and 14 is moving down theseparation channel 34 towards the waste reservoir 20. There is also aslight flow of solvent away from the injection intersection 40 towardsthe analyte and analyte waste reservoirs 16 and 18 and an appropriateflow of reagent from the reagent reservoir 22 into the separationchannel 34. The waste reservoir 20 now needs to be at the minimumpotential and the first solvent reservoir 12 at the maximum potential.All other potentials are adjusted to provide the fluid flow directionsand magnitudes as indicated in FIG. 31(b). Also, as shown in FIG. 32,the voltages applied to the solvent reservoirs 12 and 14 aremonotonically changed to move from the conditions of a large molefraction of solvent 1 to a large mole fraction of solvent 2.

At the end of the solvent programming run, the device is now ready toswitch back to the inject condition to load another sample. The voltagevariations shown in FIG. 32 are only to be illustrative of what might bedone to provide the various fluid flows in FIGS. 31(a)-(c). In an actualexperiment some to the various voltages may well differ in relativemagnitude.

While advantageous embodiments have been chosen to illustrate theinvention, it will be understood by those skilled in the art thatvarious changes and modifications can be made therein without departingfrom the scope of the invention as defined in the appended claims.

What is claimed is:
 1. A method of controlling movement of a material ina fluid in a microscale channel system, comprising providing at leastfirst, second, third and fourth fluid filled reservoirs in fluidcommunication with each other through a microscale channel system; andsimultaneously applying selected voltages at least to said first, secondand third fluid reservoirs and controlling voltage at said fourthreservoir, thereby controlling material movement in the microscalechannel system.
 2. The method of claim 1, wherein the step ofsimultaneously applying selected voltages at the first, second and thirdfluid reservoirs creates a potential difference between the first andsecond reservoirs.
 3. The method of claim 1, wherein the first, second,third and fourth reservoirs are in fluid communication at a channelintersection and the step of simultaneously applying selected voltagesat least to the first, second and third reservoirs further causes flowof material disposed in the channel intersection out of the channelintersection to at least one of the first, second, third and fourthreservoirs.
 4. The method of claim 3, further comprising the step offlowing material into the channel intersection from at least one of thefirst, second and third reservoirs prior to the step of simultaneouslyapplying selected voltages at least to the first, second and thirdreservoirs to flow material out of the channel intersection.
 5. Themethod of claim 4, wherein the step of flowing material out of thechannel intersection flows material from the channel intersection towardat least two of the first, second, third and fourth reservoirs.
 6. Themethod of claim 5, wherein the step of flowing material out of thechannel intersection flows material from the channel intersection towardat least three of the first, second, third and fourth reservoirs.
 7. Themethod of claim 1, wherein the microscale channel system comprises afirst channel intersection and a second channel intersection, first andthird channel segments being in fluid communication at the first channelintersection and second and fourth channel segments being in fluidcommunication at the second channel intersection, the first reservoirbeing connected to the first channel intersection via the first channelsegment, the second fluid reservoir being in fluid communication withthe second channel intersection by a second channel segment, the thirdfluid reservoir being connected to the first channel intersection via athird channel segment and the fourth fluid reservoir being in fluidcommunication with the second channel intersection via a fourth channelsegment, and the first and second channel intersection being connectedby a fifth channel segment.
 8. The method of claim 7, wherein the stepof simultaneously applying selected voltages at least to the first,second and third reservoirs moves a material disposed in a fluid in thefirst reservoir from the first reservoir toward the second reservoirthrough the fifth channel segment.
 9. The method of claim 7, wherein thestep of simultaneously applying selected voltages at least to the first,second and third reservoirs moves a first material disposed in a fluidin the first reservoir from the first reservoir toward the secondreservoir through the fifth channel segment, and a second materialdisposed in a fluid in the third reservoir from the third reservoir intothe fifth channel segment.
 10. The method of claim 9, whereincontrolling voltage at said fourth reservoir comprises applying avoltage at the fourth fluid reservoir, which, together with the voltagesapplied to the first, second and third fluid reservoirs, effectsmovement of a first material disposed in the first fluid filledreservoir from the first fluid filled reservoir toward the second fluidfilled reservoir, and from the third and fourth channel segments intothe fifth channel segment.
 11. The method of claim 7, wherein the stepof simultaneously applying selected voltages at least to the first,second and third reservoirs moves material disposed in the fifth channelsegment out of the fifth channel segment into at least one of the thirdand fourth channel segments.
 12. The method of claim 7, wherein the stepof simultaneously applying selected voltages at least to the first,second and third reservoirs moves material disposed in the fifth segmentout of the fifth channel segment into at least two of the first, second,third and fourth channel segments.
 13. The method of claim 7, whereinthe step of simultaneously applying selected voltages at least to thefirst, second and third reservoirs moves material disposed in the fifthchannel segment out of the fifth channel segment into the first, secondand third channel segments.
 14. The method of claim 7 further comprisingdetection a flowing material in the microscale channel system.
 15. Themethod of claim 14, wherein the detecting step comprises optical orfluorescent detection of the material in the microscale channel system.16. The method of claim 14, wherein the detecting step is carried outusing one or more of a CCD, a camera and a laser.
 17. The method ofclaim 7, further comprising detecting a flowing material in one of thethird and fourth channel segments.
 18. The method of claim 17, whereinthe detecting step comprises optical or fluorescent detection of thematerial in at least one of the third or fourth channel segments. 19.The method of claim 18, wherein the detecting steps is carried out usingone or more of a CCD camera and a laser.
 20. The method of claim 1,further comprising providing at least a fifth fluid-filled reservoir influid communication with said first, second, third and fourth fluidfilled reservoirs through the microscale channel system.
 21. The methodof claim 20, further comprising providing at least a sixth fluid-filledreservoir in fluid communication with said first, second, third andfourth fluid filled reservoirs through the microscale channel system.22. The method of claim 1, wherein the first reservoir comprises asample material, the second reservoir comprises a first buffer materialand the third reservoir comprises a second buffer material.
 23. Themethod of claim 22, wherein the first and second buffer materials arethe same.
 24. The method of claim 22, wherein the first and secondbuffer materials are different.
 25. The method of claim 1, whereincontrolling voltage at said fourth reservoir comprises applying aselected voltage at the fourth reservoir.
 26. The method of claim 25,wherein the step of simultaneously applying selected voltages to atleast one of the first, second and third reservoirs and to the fourthreservoir creates a potential difference between the first reservoir andthe second reservoir and between the first reservoir and at least one ofthe third and fourth reservoirs.
 27. The method of claim 11, whereincontrolling voltage at said fourth reservoir comprises grounding thefourth reservoir.
 28. The method of claim 1, wherein the microscalechannel system comprises a channel intersection wherein the first,second third and fourth reservoirs are in fluid communication with thechannel intersection by first, second, third and fourth channelsegments, respectively.
 29. The method of claim 1, wherein themicroscale channel system comprises a channel intersection, wherein thefirst, second, third and fourth reservoirs are connected to the channelintersection by first, second, third and fourth channel segments andsaid channel intersection being in the form of a cross.
 30. The methodof claim 1, wherein the microscale channel system comprises a channelintersection and the step of simultaneously applying selected voltagesat least to the first, second and third reservoirs flows a materialdisposed within the fluid into the microscale channel system from one ormore of the first, second and third reservoirs.
 31. The method of claim30 further comprising the step of changing said selected voltagesapplied at least to one of the first, second and third reservoirs toflow material out of the channel intersection toward at least one othersaid reservoir.